Methods and materials for making simvastatin and related compounds

ABSTRACT

The invention disclosed herein relates to methods and materials for producing simvastatin and related compounds such as huvastatin.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a divisional application that claims the benefitunder 35 U.S.C. § 121 of U.S. patent application Ser. No. 14/599,100,filed Jan. 16, 2015, which is a divisional application that claims thebenefit under 35 U.S.C. § 121 of U.S. patent application Ser. No.13/540,188, filed Jul. 2, 2012, now U.S. Pat. No. 8,951,754, which is acontinuation of U.S. patent application Ser. No. 12/227,671, filed Nov.24, 2008, now U.S. Pat. No. 8,211,664, which is the National Stage ofInternational Application No. PCT/US2007/012362 (InternationalPublication No. WO2007/139871), filed May 24, 2007, which claimspriority under Section 119(e) from U.S. provisional patent applicationNo. 60/808,088, filed May 24, 2006, the entire contents of which areincorporated herein by reference.

FIELD OF THE INVENTION

The invention relates to methods and materials for biosynthesizingcompounds such as simvastatin including procedures using microbialhosts.

BACKGROUND OF THE INVENTION

Simvastatin is a semisynthetic derivative of the natural productlovastatin, which can be isolated from the fermentation broth ofAspergillus terreus. Both lovastatin and simvastatin are cholesterollowering drugs that substantially lower the risk of heart disease amongadults. Lovastatin and simvastatin are marketed by Merck Co. as Mevacorand Zocor, respectively. Simvastatin is a more potent derivative oflovastatin and is the second best selling drug in the United States in2005, with an expect sales of $4.5 billion in the US alone.

The gene cluster for lovastatin biosynthesis in A. terreus (see, e.g.,J. Kennedy, K. et. al., Science, 1999, 284, 1368-1372; and C. R.Hutchinson, J. et. al., Antonie Van Leeuwenhoek 2000, 78, 287-295) hasbeen described previously (see, e.g., U.S. Pat. No. 6,391,583, thecontents of which are herein incorporated by reference). Encoded in thegene cluster is a 46 kD protein LovD, that was initially identified asan esterase homolog. Monacolin J, the immediate biosynthetic precursorof lovastatin, is assembled by the upstream megasynthase LovB (see,e.g., L. Hendrickson, C. R. et. al., Chem. Biol. 1999, 6, 429-439),(also known as lovastatin nonaketide synthase, LNKS), enoylreductaseLovC and CYP450 oxygenases. The five carbon unit side chain issynthesized by LovF (lovastatin diketide synthase, LDKS) throughcondensation between an acetyl-CoA and a malonyl-CoA. The condenseddiketide undergoes methylation and reductive tailoring by the individualLovF domains to yield an α-S-methylbutyryl thioester covalently attachedto the phosphopantetheine arm on the acyl carrier protein (ACP) domainof LovF (see, e.g., J. Kennedy, K. et. al., Science, 1999, 284,1368-1372 and C. R. Hutchinson, J. et. al., Antonie Van Leeuwenhoek2000, 78, 287-295), and Lovastatin is subsequently produced frommonacolin J. Inactivation of either LovD or LovF in A. terreus leads toaccumulation of the precursor monacolin J (see, e.g., J. Kennedy, K. et.al., Science, 1999, 284, 1368-1372 and C. R. Hutchinson, J. et. al.,Antonie Van Leeuwenhoek 2000, 78, 287-295).

Once lovastatin is produced via fermentation in an A. terreus host forexample, simvastatin can be produced from lovastatin. Currently,simvastatin is a semisynthetic derivative of lovastatin. Lovastatin isobtained via fermentation of the A. terreus host. After purification ofthe compound, the semisynthesis can proceed as follows: 1) the2-methylbutyrate side arm can be hydrolyzed in the presence of base toyield the intermediate monacolin J; 2) lactonize the free acid; 3) thealcohol functional group at C13 is protected with a protection group(such as tert-butyldimethylsilyl); 4) Esterification of the exposed C8alcohol with an acyl substrate such as 2-dimethylbutyryl chloride toyield a C13 protected version of simvastatin, and 5) Deprotection of C13OH to yield simvastatin (FIG. 3).

Various multistep synthesis of simvastatin have been describedpreviously (see, e.g., PCT WO 2005/066150 and U.S. Application Nos.20050080275 and 20040068123, the contents of which are hereinincorporated by reference). For example, a widely used process startswith the hydrolysis of the C8 ester in lovastatin to yield the triolmonacolin J, followed by selective silylation of the C13 alcohol,esterification of C8 alcohol with dimethylbutyryl chloride anddeprotection of C13 alcohol to yield simvastatin (see, e.g., W. F.Hoffman, et. al., J. Med. Chem. 1986, 29, 849-852). Enzymatictransformations using lipases and esterases have been investigated asalternatives to chemical derivation (see, e.g., PCT WO 2005/040107, PCTWO 94/26920 and T. G. Schimmel, et. al., Appl. Environ. Microbiol. 1997,63, 1307-1311, the contents of which are herein incorporated byreference). However, the requirement of regioselective esterificationinvariably involves protection of other alcohol groups and often leadsto lowered overall yield. Therefore, a specific reagent that is able toselectively acylate C8 of monacolin J is important towards the efficientsynthesis of simvastatin and additional statin analogs.

Variations of the above schemes are common, however, most procedureswill invariably involve isolation of lovastatin first, hydrolysis of themethylbutyrate side chain, protection of the free alcohol, reaction withan acyl substrate, and deprotection. Although the chemicaltransformations involved are relatively simple, they are inefficient andinvolve multiple steps and therefore contribute to the current high costof manufacturing simvastatin ($3 per pill).

SUMMARY OF THE INVENTION

The present invention provides methods and materials designed to takeadvantage of biological processes by which lovastatin is made in orderto produce the lovastatin derivative, simvastatin. The present inventionalso provides methods and materials designed to take advantage ofbiological processes by which lovastatin is made in order to producerelated compounds such as the pravastatin derivative, huvastatin. Asnoted above, biological processes for the production of lovastatin fromthe fermentation of A. terreus are well known in the art. In typicalprocesses for producing lovastatin, the decalin core and the HMG-CoAmoieties that mimic portions of the lovastatin compound are synthesizedby the lovastatin nonaketide synthase (LNKS) and three accessory enzymesin vivo. The 2-methylbutyrate side chain is synthesized by lovastatindiketide synthase (LDKS) in vivo. The 2-methylbutyrate is covalentlyattached to the acyl carrier domain of LovF via a thioester linkage(FIG. 6). An acyltransferase, LovD, then is able to transfer the2-methylbutyrate selectively to the C8 hydroxyl group from LDKS in onestep to yield lovastatin. As disclosed in detail below, it is nowpossible to generate simvastatin and related compounds both in vitro orin vivo by manipulating these processes.

Embodiments of the invention include methods for generating simvastatinwithout the multiple chemical synthesis steps that are currentlyemployed to generate this compound. Typical embodiments of the inventiondo not require the purification of lovastatin as a first step, followedby the further semisynthetic procedures and instead use a singlefermentation step to produce simvastatin. The processes disclosed hereinare designed so that fermentation facilities currently producinglovastatin can be converted to producing simvastatin and relatedcompounds with minimal modifications. The materials used in theprocesses disclosed herein are relatively inexpensive and thepurification steps are well known in the art and easily practiced.

Those of skill in the art will understand that the disclosure providedherein allows artisans to produce a wide variety of embodiments of theinvention. A typical embodiment of the invention is a method of makingsimvastatin by combining together monacolin J; an acyl thioester thatdonates an acyl moiety to the C8 hydroxyl group of monacolin J in thepresence of LovD acyltransferase; and LovD acyltransferase; and thenallowing the LovD acyltransferase use an acyl group from the acylthioester to regioselectively acylate the C8 hydroxyl group of monacolinJ; so that simvastatin is made. In typical embodiments of the invention,the LovD acyltransferase has the amino acid sequence of SEQ ID NO: 1 orSEQ ID NO: 2. A related embodiment of the invention is a method ofmaking simvastatin comprising the steps of combining togetherlovastatin; an acyl thioester that donates an acyl moiety to the C8hydroxyl group of monacolin J in the presence of LovD acyltransferase;and LovD acyltransferase. In this embodiment of the method, the LovDacyltransferase is then allowed to hydrolyze lovastatin into monacolinJ; and to then use an acyl group from the acyl thioester toregioselectively acylate the C8 hydroxyl group of monacolin J; so thatsimvastatin is made. In certain embodiments, the simvastatin is made invitro in the absence of an isolated organism.

As is discussed in detail below, the methods and materials of theinvention that are used to make simvastatin can be adapted to producecompounds that are structurally similar to simvastatin, for examplehuvastatin. In this context, one embodiment of the invention is a methodof making huvastatin comprising the steps of combining togetherhydrolyzed pravastatin tetra-ol; an acyl thioester that donates an acylmoiety to the C8 hydroxyl group of hydrolyzed pravastatin tetra-ol inthe presence of LovD acyltransferase; and LovD acyltransferase; and thenallowing the LovD acyltransferase use an acyl group from the acylthioester to regioselectively acylate the C8 hydroxyl group ofhydrolyzed pravastatin tetra-ol, so that huvastatin is made. A relatedembodiment of the invention is a composition of matter comprising:hydrolyzed pravastatin tetra-ol; an acyl thioester that donates an acylmoiety to the C8 hydroxyl group of hydrolyzed pravastatin tetra-ol inthe presence LovD acyltransferase; LovD acyltransferase; and huvastatin.Consequently, embodiments of the invention include processes for makingsimvastatin or huvastatin composition of matter substantially as hereindisclosed and exemplified.

In some embodiments of the invention, the monacolin J; the acylthioester and the LovD acyltransferase are combined in a fermentationmedia in the presence of an isolated organism that produces the LovDacyltransferase and further wherein the organism is EscherichiaAspergillus terreus, Monascus ruber, Monascus purpureus, Monascuspilosus, Monascus vitreus, Monascus pubigerus, Candida cariosilognicola,Aspergillus ogea, Doratomyces stemonitis, Paecilomyces virioti,Penicillum citrinum, Penicillin chgsogenum, Scopulariopsis brevicaulisor Trichoderma viride. Optionally, the isolated organism is Aspergillusterreus that expresses LovD polypeptide of SEQ ID NO: 1. In certainembodiments, the Aspergillus terreus does not express LovF polypeptideof SEQ ID NO: 3. Alternatively, the organism can be Escherichia colithat expresses LovD polypeptide of SEQ ID NO: 1 or SEQ ID NO: 2. Incertain embodiments, the Escherichia coli does not express bioHpolypeptide of SEQ ID NO: 4. As discussed in detail below, the isolatedorganism can be grown under one of a variety of fermentation conditionsknown in the art and the exact conditions can be selected for examplebased upon the fermentation requirements of specific organism used. Intypical embodiments of the invention, the organism is grown at atemperature between 30-40° C., for a time period between at least 4 toat least 48 hours and at a pH between 6.5-8.5. In illustrativeembodiments, the organism is grown in a fermentation media comprisingLB, F1 or TB media.

Optionally, the monacolin J that is combined with the other constituentsin the methods of the invention is produced by an isolated organismwithin the fermentation media, for example one of the organisms listedabove that also produces the LovD acyltransferase. Alternatively, themonacolin J that is combined with the other constituents in the methodsof the invention is produced by a different organism that produces thiscompound that is added to the fermentation media and grows along withthe organism that produces the LovD acyltransferase. In this context anumber of organisms known in the art to produce monacolin J can beadapted for use with these embodiments of the invention (see, e.g. Endoet al., J Antibiot (Tokyo). 1985 March; 38(3):420-2. And Kennedy et al.,1999 May 21; 284(5418):1368-72). In another embodiment of the invention,monacolin J is derived from an alternative exogenous source (e.g. achemical synthesis process) and added to the fermentation mixture.

In typical embodiments of the invention, acyl thioester that can donatean acyl moiety to the C8 hydroxyl group of monacolin J in the presenceof LovD acyltransferase is derived from an exogenous source (e.g. achemical synthesis process) and added to the fermentation mixture. Avariety of such acyl thioesters are disclosed herein. Typically the acylthioester is a butyrlyl-thioester, a N-acetylcysteamine thioester or amethyl-thioglycolate thioester. Optionally, the acyl thioester comprisesmedium chain length (C3-C6) acyl group moieties. In certain embodimentsof the invention, the acyl thioester is able to cross the cellularmembranes of Escherichia coli or Aspergillus terreus cells growingwithin a fermentation media. Typically, the acyl thioester is selectedfrom the group consisting ofα-dimethylbutyryl-S-methyl-mercaptopropionate (DMB-S-MMP),dimethylbutyryl-S-ethyl mercaptopropionate (DMB-S-EMP) anddimethylbutyryl-S-methyl thioglycolate (DMB-S-MTG) anddimethylbutyryl-S-methyl mercaptobutyrate (DMB-S-MMB). In anillustrative embodiment, the acyl thioester isα-dimethylbutyryl-S-methyl-mercaptopropionate that is combined infermentation media in a concentration range of 1 mM-100 mM.

In certain embodiments of the invention, the method results in at least95%, 96%, 97%, 98% or 99% of the monacolin J added to the combinationbeing converted to simvastatin. Optionally, the method of the inventionproduces a composition of matter comprising 0%-1% of the monacolin Jthat was initially added to the combination. Certain embodiments of themethods for making simvastatin and related compounds include furthersteps to purify these compounds. For example, embodiments of theinvention can include at least one purification step comprising lysis ofcells of an isolated organism present in the combination. Embodimentscan also include at least one purification step comprisingcentrifugation of cells or cell lysates of an isolated organism presentin the combination. Moreover, embodiments can include at least onepurification step comprising precipitation of one or more compoundspresent in the combination. One embodiment of a precipitation stepcomprises the precipitation of a free acid form of simvastatin.Optionally in such embodiments, one can then convert this free acid formof simvastatin to a simvastatin salt. Embodiments of the invention canalso include at least one purification step comprising filtration of oneor more compounds present in the combination. In addition, embodimentscan include at least one purification step comprising a high performanceliquid chromatography (HPLC) analysis of one or more compounds presentin the combination.

Embodiments of the invention include compositions of matter useful formaking and/or made by the processes disclosed herein. For example, oneembodiment of the invention is a composition of matter comprising:monacolin J; an acyl thioester that donates an acyl moiety to the C8hydroxyl group of monacolin J in the presence LovD acyltransferase; LovDacyltransferase; and simvastatin. Optionally, the composition furthercomprises an isolated organism such as Escherichia coli, Aspergillusterreus, Monascus ruber, Monascus purpureus, Monascus pilosus, Monascusvitreus, Monascus pubigerus, Candida cariosilognicola, Aspergillus ogea,Doratomyces stemonitis, Paecilomyces virion, Penicillum citrinum,Penicillin chysogenum, Scopulariopsis brevicaulis or Trichodermavirioti. In typical embodiments, the organism in the composition isAspergillus terreus or Escherichia coli that expresses LovD polypeptideof SEQ ID NO:1. In one embodiment of the invention, the organism isAspergillus terreus that does not express LovF polypeptide of SEQ ID NO:3. In another embodiment of the invention, the organism is Escherichiacoli that does not express bioH polypeptide of SEQ ID NO: 4. In certainembodiments of the invention, isolated organism within the compositionhas been transduced with an expression vector encoding Aspergillusterreus LovD (SEQ ID NO: 1).

A variety of acyl thioesters that can be used in the compositions of theinvention are disclosed herein. Typically the acyl thioester is abutyrlyl-thioester, a N-acetylcysteamine thioester or amethyl-thioglycolate thioester. Optionally, the acyl thioester comprisesmedium chain length (C3-C6) acyl group moieties. In certain embodimentsof the invention, the acyl thioester is able to cross the cellularmembranes of Escherichia coli or Aspergillus terreus cells growingwithin a fermentation media. Typically, the acyl thioester is selectedfrom the group consisting ofα-dimethylbutyryl-S-methyl-mercaptopropionate (DMB-S-MMP),dimethylbutyryl-S-ethyl mercaptopropionate (DMB-S-EMP) anddimethylbutyryl-S-methyl thioglycolate (DMB-S-MTG) anddimethylbutyryl-S-methyl mercaptobutyrate (DMB-S-MMB). In anillustrative embodiment, the acyl thioester isα-dimethylbutyryl-S-methyl-mercaptopropionate that is combined infermentation media in a concentration range of 1 mM-100 mM. In someembodiments of the invention, the composition further compriseslovastatin and the amount of simvastatin in the composition is greaterthan the amount of lovastatin in the composition.

In certain embodiments of the invention, further components and/ormethodological steps can be combined with one or more of the methods andmaterials discussed above. For example, the methods can further compriseusing high cell-density fermentation to increase the effectiveconcentration of LovD acyltransferase and optimise fermentationconditions or increasing LovD acyltransferase catalytic efficienciestowards the one or more thioesters protein engineering. Many othercomponents or methods can be used to increase the production ofsimvastatin or of an intermediary or related compound that facilitatesthe production of simvastatin.

Embodiments of the invention also include articles of manufacture and/orkits designed to facilitate the methods of the invention. Typically suchkits include instructions for using the elements therein according tothe methods of the present invention. Such kits can comprise a carriermeans being compartmentalized to receive in close confinement one ormore container means such as vials, tubes, and the like, each of thecontainer means comprising one of the separate elements to be used inthe method. One of the containers can comprise a vial, for example,containing LovD acyltransferase and/or A. terreus and another vialcontaining an thioester compound or the like, both of which can be addedto a fermentation mixture to produce simvastatin and/or huvastatin orthe like.

Additional embodiments of the invention are discussed below.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. A. Lovastatin. B. Simvastatin. The two compounds differ by onemethyl substitution at the α position.

FIG. 2. Lovastatin (1), Simvastatin (2), Pravastatin (5), Huvastatin (6)and related compounds

FIG. 3. Current method of manufacturing simvastatin.

FIG. 4. Acyl transfer reaction of the invention in A. terreus. Serine 76can be the active site nucleophile.

FIG. 5. A) HPLC (238 nm) trace showing formation of 4 by LovD mediatedacyl transfer. Gradient: 60% B-95% B, 5 min; 95% B, 15 min; A: H₂O+0.1%TFA; B: acetonitrile+0.1% TFA; a) LovD+monacolin J+butyryl-CoA timecourse (0, 1, 3, 6 and 10 hours); b) LovD S76A+monacolin J+butyryl-CoA,10 hrs; c) LovD+butyryl-SNAC, 10 hrs; d) LovD+butyryl-SMTG, 10 hrs.Assay conditions: 1 mM monacolin J, 4 mM butyryl-CoA, 10 μLovD, 50 mMHEPES, pH 7.9, 25 degrees C. (B) Conversion as a function of time for(⋄) butyryl-CoA (♦) butyryl-SNAC, (▪) butyryl-SMTG. The apparent k_(cat)values reported throughout the text are initial turnover rates in thelinear range. (C) Michaelis-Menten plot of LovD catalyzed hydrolysis oflovastatin to monacolin J. The reaction progress is monitored by HPLC.k_(cat): 0.21±0.01 min⁻¹; Kin: 0.56±0.05 mM.

FIG. 6. LovD catalyzes the acyltransfer to yield lovastatin. The acyldonor is attached to LDKS.

FIG. 7. Acyl-SNAC. The shaded circle denotes any functional group.

FIG. 8. HPLC (238 nm) trace showing formation of simvastatin andlovastatin by LovD mediated acyl transfer. Gradient is the same as thatdescribed in FIG. 1; a) authentic standards of lovastatin andsimvastatin; b) monacolin J+α-S-methylbutyryl-SNAC; c) monacolinJ+α-dimethylbutyryl-SNAC. Assay conditions: 1 mM monacolin J, 10 mMacyl-SNAC, 100 μM LovD, 50 mM HEPES, pH 7.9, 25° C., 6 hrs.

FIG. 9. HPLC (238 nm) traces showing formation of pravastatin andhuvastatin by LovD mediated acylation of the tetra-ol 7. Gradient: 5%B-95% B, 5 min; 95% B, 15 min; A: H₂O+0.1% TFA; B: acetonitrile+0.1%TFA; a) authentic standards of pravastatin and 7; b)7+α-S-methylbutyryl-SNAC; c) 7+α-dimethylbutyryl-SNAC. Assay conditions:1 mM monacolin J, 10 mM acyl-SNAC, 100 uM LovD, 50 mM HEPES, pH 7.9, 25degrees C., 6 hrs.

FIG. 10. Microbial conversion of monacolin J into simvastatin.

FIG. 11. LovD catalyzed hydrolysis and LovD catalyzed acylation. FIG. 11encompasses embodiments of the invention including compositions ofmatter that produces huvastatin or simvastatin in the presence of LovDand an acyl donor. FIG. 11 also illustrates a method of producingsimvastatin comprising hydrolyzing lovastatin in the presence of LovD toproduce monacolin J. Monacolin J is thereby converted to simvastatin inthe presence of an acyl donor. Likewise, FIG. 11 also illustrates amethod of producing huvastatin comprising hydrolyzing pravastatin in thepresence of LovD to produce hydrolyzed pravastatin. Hydrolyzedpravastatin is thereby converted to huvastatin in the presence of anacyl donor.

FIG. 12. Production of huvastatin in a microorganism expressing LovD.FIG. 12 provides illustrations of embodiments of the invention includingthe production of huvastatin in an organism expressing LovD.

FIG. 13. (A) Whole cell biocatalytic conversion of simvastatin acid frommonacolin J acid and DMB-S-MMP. The E. coli strain overexpresses theacyltransferase LovD. Hydrolysis of DMB-S-MMP to yield DMB-S-MPA is acompeting reaction. (B) top trace: typical profile of reactants andproducts from the whole cell conversion experiment. Bottom traces:standards for DMB-S-MMP and DMB-S-MPA. The spectra were collected at 238nm.

FIG. 14. (A) Thin layer chromatography showing the organic extracts ofsixty E. coli strains supplemented with DMB-S-MMP. The strainsidentifications can be found in Table 2. The TLC plate was developedwith 20% EA in hexane to separate DMB-S-MMP (top) and DMB-S-MPA(bottom). The middle spot is an unknown compound. In all strains except#56, DMB-S-MMP was hydrolyzed to DMB-S-MPA after 10 hours of incubation.The ΔbioH strain did not hydrolyze DMB-S-MMP. (B) Confirmation of TLCresults using HPLC. No DMB-S-MPA can be detected in the extract of ΔbioHstrain. The spectra were collected at 234 nm.

FIG. 15. (A) Michaelis-Menten kinetics of BioH towards DMB-S-MMP. Theresults are averaged for three runs. The kcat and Km values are 260±45sec-1 and 229±26 μM, respectively. The high standard deviation observedat high DMB-S-MMP concentration may be due to the poor solubility of thesubstrate in buffer (50 mM HEPES, pH 7.9, 10% DMSO). (B) Comparison ofhydrolysis rates for three different dimethylbutyryl thioesters. Thereactions were performed with 1 mM thioester and 10 nM BioH.

FIG. 16. (A) Comparison of growth rates and final cell density forBL21(DE3)/pAW31 (grey 0) and YT2/pAW31 (without biotin: 0; with 0.15mg/L biotin: ●) in F1 minimal media. Cells were grown to OD600 ˜0.5,induced with 100 μM IPTG and shifted to a 20° C. shaker for 12 hours.The ΔbioH strain YT2 requires addition of exogenous biotin to maintainrobust cell growth in synthetic medium. (B) Comparison of simvastatinconversion as a function of time (15 mM MJ, 25 mM DMB-S-MMP). YT2/pAW31(∘) is significantly faster than BL21(DE3)/pAW31 (●) (data taken fromXie et al., (2007) Appl Environ Microbiol 73: 2054-2060) in reaching 99%conversion. Both strains displayed a lag phase immediately followingsubstrate addition (0 hour) and a lag phase near reaction completion.The reaction velocities between the lag phases are linear. Theconversion rate for YT2/pAW31 (1.5 mM/hr) is twice as fast as that forBL21(DE3) (0.75 mM/hr)

FIG. 17. HPLC analysis (238 nm) of simvastatin purification steps.Simvastatin acid is lactonized to simvastatin prior to injection. Tracea: crude extract (combined culture broth and cell extract) of YT2/pAW31after completion of reaction (99% MJ acid conversion to simvastatinacid). The peak at 6.80 is the DMB-S-MMP; Trace b: Crude sample afterwashing with equal volume of hexane to remove unreacted DMB-S-MMP andprecipitation; Trace c: Purified simvastatin after washing with dH2O andsolubilization in ACN. The small peak seen at 6.7 minutes is simvastatinacid that is not completely lactonized (˜1%).

FIG. 18. The chemical structures of lovastatin acid, monacolin J acidand simvastatin acid. The biocatalytic reaction studied is the enzymaticconversion of monacolin J to simvastatin (block arrow). LovD is able toregioselectively acylate the C8 hydroxyl group. Two commonly usedsemisynthetic processes are shown in dashed arrows.

FIGS. 19A and 19B. Kinetic analysis of LovD catalyzed acylation ofmonacolin J to yield simvastatin using DMB-S-MMP as the acyl thioester.The y-axis is expressed as catalytic turnover (V/Eo) (FIG. 19A)Michaelis-menten kinetics of LovD as a function of monacolin Jconcentration, at a fixed DMB-SMMP concentration of 2 mM. No substrateinhibition is observed. Km (monacolin J)=0.78±0.12 mM. (FIG. 19B)Michaelis-menten kinetics of LovD as a function of DMB-S-MMPconcentration, at a fixed monacolin J concentration of 2 mM. Km(DMB-S-MMP)=0.67±0.04 mM. In both assays, the kcat is estimated to be0.66±0.03 min-1.

FIGS. 20A and 20B. Low density fermentation and biocatalysis. The E.coli strain BL21(DE3)/pAW31 expressing LovD overnight was concentrated10× to a final OD600 of 22. The substrates monacolin J and DMB-S-MMPwere added to a final concentrate of 15 and 40 mM, respectively. Theconversion was followed as function of time by HPLC analysis. (FIG. 20A)HPLC traces of the time course study. The labeled peaks are 1: monacolinJ (lactonized form); 2: DMB-S-MPA as a result of DMB-S-MMP hydrolysis;3: DMB-S-MMP; and 4: simvastatin (lactonized form). (FIG. 20B)Conversion of monacolin J to simvastatin as a function of time. Finalconversion at 24 hours was 99%. The data points are averaged values oftwo runs.

FIGS. 21A and 21B. High density fermentation and biocatalysis. (FIG.21A) Fed-batch fermentation (500 mL) with F1 minimal medium. At OD600(circles) of 5.93 (I), the temperature of the fermentation is decreasedto and maintained at RT. IPTG is added to a final concentration of 200μM and the feeding is initiated and maintained at 0.08 mL/min. Theeffective concentrations of LovD at different points of the fermentationare measured (squares). (FIG. 21B) The rate of conversion of monacolin Jto simvastatin by cells at four different points during the fermentation(indicated by 1, 2, 3 and 4) in FIG. 21A. The cells are either made“resting” by shifting to 50 mM HEPES, pH 7.9, or “non-resting” withoutmedium change.

DETAILED DESCRIPTION OF THE INVENTION

The techniques and procedures described or referenced herein aregenerally well understood and commonly employed using conventionalmethodology by those skilled in the art, such as, for example, thewidely utilized molecular cloning methodologies described in Ausubel etal., Current Protocols in Molecular Biology, Wiley IntersciencePublishers, (1995). As appropriate, procedures involving the use ofcommercially available kits and reagents are generally carried out inaccordance with manufacturer defined protocols and/or parameters unlessotherwise noted. Unless otherwise defined, all terms of art, notationsand other scientific terminology used herein are intended to have themeanings commonly understood by those of skill in the art to which thisinvention pertains. In some cases, terms with commonly understoodmeanings are defined herein for clarity and/or for ready reference, andthe inclusion of such definitions herein should not necessarily beconstrued to represent a substantial difference over what is generallyunderstood in the art.

The definitions of certain terms are provided below.

“Lovastatin” (Mevacor) is a fungal polyketide produced by Aspergillusterreus (see, e.g., A. W. Alberts, J. et. al., Proc. Natl. Acad. Sci.U.S.A, 1980, 77, 3957-3961 and A. Endo, J. Antibiot. 1980, 33, 334-336;and J. K. Chan, et. al., J. Am. Chem. Soc. 1983, 105, 3334-3336; Y.Yoshizawa, et. al., J. Am. Chem. Soc. 1994, 116, 2693-2694). It is apharmaceutically important compound because of its potent inhibitoryactivities towards hydroxymethylglutaryl coenzyme A reductase (HMGR),the rate-limiting step of cholesterol biosynthesis, and therefore it iswidely used in the treatment of hyperlipidemia, hypercholesterolemia,and the like. Lovastatin is also referred to as Mevacor.

“Simvastatin” is an analog of lovastatin. It is favored over lovastatinbecause of the absence of adverse side effects and its highabsorbability in the stomach. Also, it has been reported thatsimvastatin prevents and reduces the risk of Alzheimer's disease (AD) byretarding the production of Ab42, β-amyloid protein associated with AD.It is known in the art that simvastatin can be synthetically prepared byway of direct methylation of the 8′-methylbutyryloxy side chain oflovastatin of formula using a methyl halide in the presence of a metalamide base. The C-methylation step has to be carried out at extremelylow temperatures (−75 to −30°) using a strong base under anhydrouscondition which is difficult to handle in mass production (see, e.g.,U.S. Pat. Nos. 5,393,893, 4,582,915, 5,763,646, 5,763,653, EP Patent No.299,656 and International Patent Publication No. WO 99/45003, thecontents of which are herein incorporated by reference). Other methodsof synthetically producing simvastatin is also known in the art. Forexample, lovastatin can be hydrolyzed with an excessive amount oflithium hydroxide to remove the 2-methylbutyryl side chain and tosimultaneously open its 6-membered lactone ring to produce a triol acid.The triol acid compound can then be heated to obtain a diol lactone. Thehydroxy group on the lactone ring of the diol lactone can be protectedto obtain a tert-butyldimethylsilyl ether and then the hydroxy group atC8 of the hexahydronaphthalene ring system can be acylated with2,2-dimethylbutaonic acid in the presence of dicyclohexyl carbodiimide,or 2,2-dimethyl chloride to produce a compound. The t-butyldimethylsilylprotecting group of the compound can then be removed in the final stepusing tetrabutylammonium fluoride to produce simvastatin (see, e.g.,U.S. Pat. No. 4,444,784, the contents of which are herein incorporatedby reference). “Lovastatin derivatives” as used herein compriseslovastatin derivatives or precursors for example pravastatin,huvastatin, simvastatin, or hydrolyzed pravastatin tetra-ol. “MonacolinJ variants” refers to monacolin J variants disclosed in the art, forexample hydrolyzed pravastatin tetra-ol or6-hydroxyl-6-desmethylmonacolin J and the like. In certain embodimentsof the invention, “Monacolin J variants” refers to Monacolin J compoundshaving substitutions at the C6 position in FIG. 2. In describingcompounds such as simvastatin, pravastatin, monacolin J and variantsetc., those of skill in the art understand that this language isintended to encompass these compounds as well as the salts of thesecompounds (e.g. pharmaceutically acceptable salts known in the art). Forexample, as is known in the art, simvastatin can occur both a free acidform as well as a simvastatin sodium, potassium or ammonium salts, andother salts derived from alkaline earth elements or other metallicsalts.

“Aspergillus terreus” or “A. terreus” is a filamentous ascomycetecommonly found in soil. A variety of A. terreus strains are know in theart, for example those deposited as, e.g., ATCC 20542 and ATCC 20541.

As is known in the art, genes related to biosynthesis of secondarymetabolites of filamentous fungi can form a cluster on the fungal genomeand are referred to as “gene clusters.” For example,“Lovastatin-producing gene cluster” can refer to a set of genes thatproduce lovastatin, the set of genes comprising, LovA, a P450I; LovC, adehydrogenase; LovD, an esterase and acyltransferase; and LovF, a ScPKSor LDKS. It has been determined previously that each of these four genes(LovA, LovC, LovD, and LovF) is required for lovastatin synthesis (see,e.g., U.S. Pat. No. 6,943,017, the contents of which are hereinincorporated by reference). LovF (LDKS gene) is characterized as apolyketide synthase gene. LovD is a putativeesterase/carboxypeptidase-like gene. Disruption of the LovF gene hasbeen done previously (see, e.g., U.S. Pat. No. 6,943,017, the contentsof which are herein incorporated by reference). LovD interacts with LovFto produce lovastatin; however, the LovD-LovF interaction is notrequired for the production of simvastatin. Moreover, another gene inthe lovastatin-producing gene cluster is LovE, which is a Zn finger thatcan regulate the transcription of the other genes. Thelovastatin-producing gene cluster also comprises LovB (NPKS gene).

“LDKS” or “LDKS gene” refers to the protein encoded by the LovF gene, amember of the lovastatin-producing gene cluster. LDKS stands forlovastatin diketide synthase. LovF is the gene that produces LDKS. LovFis also the gene that produces LovF protein. “LDKS gene” also refers tothe gene that produces LDKS. In the synthesis of lovastatin, LDKSsynthesizes the five carbon unit side chain of monacolin J throughcondensation between an acetyl-CoA and a malonyl-CoA. The condenseddiketide undergoes methylation and reductive tailoring by the individualLovF domains to yield an α-S-methylbutyryl thioester covalently attachedto the phosphopantetheine arm on the acyl carrier protein (ACP) domainof LovF.

“LovD acyltransferase” as used herein refers to those polypeptides suchas the A. terreus LovD polypeptide (e.g. SEQ ID NO: 1) that can use aacyl thioester to regiospecifically acylate the C8 hydroxyl group ofmonacolin J so as to produce simvastatin. As also disclosed herein, thisLovD enzyme can further utilize a acyl thioester to regiospecificallyacylate the C8 hydroxyl group of hydrolyzed pravastatin tetra-ol so asto produce huvastatin.

LovD acyltransferases include homologous enzymes to A. terreus LovDpolypeptide (e.g. SEQ ID NO: 1) that can be found in for example, butnot limited to, fungal polyketide gene clusters. For example, the artprovides evidence that Mlc in the compactin biosynthetic pathwaycatalyzes the identical transacylation reaction (see, e.g., Y. Abe, T.et. al., Mol Genet Genomics. 2002, 267, 636-646), whereas anacyltransferase in the squalestatin pathway can catalyze a similarreaction between an ACP-bound tetraketide thioester and an aglycon (see,e.g., R. J. Cox, F. et. al., Chem Commun (Camb) 2004, 20, 2260-2261).The amino acid sequence of A. terreus LovD polypeptide (e.g. SEQ IDNO: 1) resembles type C β-lactamase enzymes, which catalyze thehydrolytic inactivation of the β-lactam class of antibiotics (see.,e.g., E. Lobkovsky, E. M. et. al., Biochemistry, 1994, 33, 6762-6772 andA. Dubus, D. et. al., Biochem. J. 1993, 292, 537-543). Alignment of A.terreus LovD polypeptide (e.g. SEQ ID NO: 1) with the Enterobactercloacae P99 lactamse (see, e.g., S. D. Goldberg, et. al., Protein Sci.2003, 12, 1633-1645) shows moderate sequence homology, includingpotentially conserved active site residues, such as the catalytic Ser76,Lys79, Tyr188, and Lys315 (see. e.g. S. D. Goldberg, et. al., ProteinSci. 2003, 12, 1633-1645).

LovD acyltransferases can also refer to both genetically engineered andnaturally occurring enzymes that are related to A. terreus LovDpolypeptide (e.g. SEQ ID NO: 1) in sequence but containing slight aminoacid differences (e.g. 1-10 amino acid substitution mutations).Simvastatin, for example, can be produced from naturally occurringenzymes that are similar to A. terreus LovD polypeptide (e.g. SEQ IDNO: 1) in sequence (e.g. the MlCH from the compactin cluster). “LovDacyltransferases” can also refer to mutants of A. terreus LovDpolypeptide (SEQ ID NO: 1). It is known in the art that mutants can becreated by standard molecular biology techniques to produce, forexample, mutants of SEQ ID NO: 1 that improve catalytic efficiencies orthe like. For example, we are currently using rational and directedevolution approaches to improve the catalytic turnover rates of A.terreus LovD. Typically such mutants will have a 50%-99% sequencesimilarity to SEQ ID NO: 1. In this context, the term “LovD homologousenzyme” includes a LovD polypeptide having at least 80%, 85%, 90%, 95%,97%, 98% or 99% sequence identity with the amino acid sequence set outin SEQ ID NO: 1, wherein the polypeptide has the ability to utilize aacyl thioester to regiospecifically acylate the C8 hydroxyl group ofmonacolin J so as to produce simvastatin and/or utilize a acyl thioesterto regiospecifically acylate the C8 hydroxyl group of hydrolyzedpravastatin tetra-ol so as to produce huvastatin. Such mutants arereadily made and then identified in assays which observe the productionof a desired compound such as simvastatin (typically using A. terreusLovD polypeptide (e.g. SEQ ID NO: 1) as a control). These mutants can beused by the methods of this invention to make simvastatin or huvastatin,for example.

“Heterologous” as it relates to nucleic acid sequences such as codingsequences and control sequences denotes sequences that are not normallyassociated with a region of a recombinant construct, and/or are notnormally associated with a particular cell. Thus, a “heterologous”region of a nucleic acid construct can be an identifiable segment ofnucleic acid within or attached to another nucleic acid molecule that isnot found in association with the other molecule in nature. For example,a heterologous region of a construct can include a coding sequenceflanked by sequences not found in association with the coding sequencein nature. Similarly, a host cell transformed with a construct, which isnot normally present in the host cell, would be considered heterologous(see, e.g., U.S. Pat. Nos. 5,712,146 6,558,942, 6,627,427, 5,849,541 thecontents of which are herein incorporated by reference). For instance, aconstruct with Lov genes can be isolated and expressed in non-lovastatinproducing fungi or yeast host cells, and lovastatin can thereby beproduced (see, e.g., U.S. Pat. Nos. 6,391,583 and 6,943,017, thecontents of which are herein incorporated by reference). As anotherexample, prokaryotes such as bacteria can be host cells also, as isknown in the art. Fungal genes may also be cloned into an expressionvector for expression in prokaryotes (see, e.g., U.S. Pat. No.5,849,541, the contents of which are herein incorporated by reference).

A prokaryote such as E. coli can be used as a heterologous host. Aplasmid can be constructed with a gene of interest and the plasmid canbe transformed into E. coli. The gene of interest can be translated andthe protein derived from the gene of interest can be purifiedthereafter. This method of expression and protein purification is knownin the art. For example, LovD exons from A. terreus can be individuallyamplified from the genomic DNA of A. terreus and spliced to yield acontinuous open reading frame using splice overlap extension PCR.Restriction sites can be introduced, and the gene cassette can beligated to a vector to yield an expression construct that can betransformed into E. coli. Thereby, E. coli can be used as a heterologoushost for expression of A. terreus genes. E. coli can be co-cultured withanother strain that produces another substrate of interest.Additionally, substrates can be added to this culture or co-culture.Heterologous expression of the lovastatin biosynthesis genes is known inthe art (see, e.g., U.S. Pat. Nos. 6,391,583 and 6,943,017, the contentsof which are herein incorporated by reference).

As another example, certain polyketides, such as polyketides from fungi,or other organisms, can be heterologously expressed in E. coli, yeast,and other host organisms. These host organisms can be supplemented withother substrates, since they can require both the heterologousexpression of a desired PKS and also the enzymes that produce at leastsome of the substrate molecules required by the PKS (see, e.g., U.S.Pat. No. 7,011,959, the contents of which are herein incorporated byreference). Similarly, fungal Lov genes can be expressed in E. coli orother bacterium, and these host bacteria can be supplemented with othersubstrates, such as acyl-SNAC or other acyl donor groups. These acyldonor groups can be cell permeable, and enter the bacterial cell.

“Expression vector” refers to a nucleic acid that can be introduced intoa host cell. As is known in the art, an expression vector can bemaintained permanently or transiently in a cell, whether as part of thechromosomal or other DNA in the cell or in any cellular compartment,such as a replicating vector in the cytoplasm. An expression vector alsocomprises a promoter that drives expression of an RNA, which typicallyis translated into a polypeptide in the cell or cell extract. Forexample, suitable promoters for inclusion in the expression vectors ofthe invention include those that function in eukaryotic or prokaryotichost cells. Promoters can comprise regulatory sequences that allow forregulation of expression relative to the growth of the host cell or thatcause the expression of a gene to be turned on or off in response to achemical or physical stimulus. For E. coli and certain other bacterialhost cells, promoters derived from genes for biosynthetic enzymes,antibiotic-resistance conferring enzymes, and phage proteins can be usedand include, for example, the galactose, lactose (lac), maltose,tryptophan (trp), beta-lactamase (b/a), bacteriophage lambda PL, and T5promoters. In addition, synthetic promoters, such as the tac promoter(U.S. Pat. No. 4,551,433), can also be used. For E. coli expressionvectors, it is useful to include an E. coli origin of replication, suchas from pUC, p1P, p1I, and pBR. For efficient translation of RNA intoprotein, the expression vector also typically contains aribosome-binding site sequence positioned upstream of the start codon ofthe coding sequence of the gene to be expressed. Other elements, such asenhancers, secretion signal sequences, transcription terminationsequences, and one or more marker genes by which host cells containingthe vector can be identified and/or selected, may also be present in anexpression vector. Selectable markers, i.e., genes that conferantibiotic resistance or sensitivity, can be used and confer aselectable phenotype on transformed cells when the cells are grown in anappropriate selective medium. For example, an expression vectorcontaining the Lov gene cluster or portions thereof can be introducedinto a heterologous host, such as E. coli. Thus, recombinant expressionvectors can contain at least one expression system, which, in turn, canbe composed of at least a portion of Lov and/or other biosynthetic genecoding sequences operably linked to a promoter and optionallytermination sequences that operate to effect expression of the codingsequence in compatible host cells.

A “coding sequence” can be a sequence which “encodes” a particular gene,such as a gene from the Lov gene cluster, for example. A coding sequenceis a nucleic acid sequence which is transcribed (in the case of DNA) andtranslated (in the case of mRNA) into a polypeptide in vitro or in vivowhen placed under the control of appropriate regulatory sequences. Theboundaries of the coding sequence are determined by a start codon at the5′ (amino) terminus and a translation stop codon at the 3′ (carboxy)terminus. A transcription termination sequence will usually be located3′ to the coding sequence.

DNA “control sequences” refer collectively to promoter sequences,ribosome binding sites, polyadenylation signals, transcriptiontermination sequences, upstream regulatory domains, enhancers, and thelike, which collectively provide for the transcription and translationof a coding sequence in a host cell.

“Operably linked” refers to an arrangement of elements wherein thecomponents so described are configured so as to perform their usualfunction. Thus, control sequences operably linked to a coding sequenceare capable of effecting the expression of the coding sequence. Thecontrol sequences need not be contiguous with the coding sequence, solong as they function to direct the expression thereof.

“Lovastatin-producing organism” refers to the wide variety of differentorganisms known in the art to produce lovastatin. These organisms thatproduce lovastatin can modified to produce simvastatin by the methods ofthis invention. A. terreus is an example of a lovastatin producingorganism. Microorganisms other than A. terreus reported to producelovastatin (mevinolin) include Monascus species, for example M. ruber,M. purpureus, M. pilosus, M. vitreus, M. pubigerus, as well asPenicillium, Hypomyces, Doratomyces, Phoma, Eupenicillium, Gymnoascus,and Trichoderma species, Pichia labacensis, Candida cariosilognicola,Aspergillus ogea, Doratomyces stemonitis, Paecilomyces virioti,Penicillum citrinum, Penicillin chysogenum, Scopulariopsis brevicaulisand Trichoderma viride (see, e.g., U.S. Pat. No. 6,391,583; Juzlova etal., J. Ind. Microbiol. 16:163-170; Gunde-Cimerman et al., FEMSMicrobiol. Lett. 132:39-43 (1995); and Shindia et al., Folio Microbiol.42:477-480 (1997), the contents of which are herein incorporated byreference).

“Non-lovastatin-producing organisms” as used herein refers to a numberorganisms that do not produce lovastatin absent manipulation by man(e.g. E. Coli). These organisms can be induced to produce LovD, orcultured in the presence of LovD to produce lovastatin or simvastatin bythe methods of this invention, for example.

“A. terreus having a disruption in the LDKS gene” comprises an A.terreus without the LDKS gene, having a LDKS gene that is mutated,having a LDKS gene that is knocked-out, having a LDKS gene that isdeleted, having a LDKS gene whose expression is disrupted, or having aLDKS gene that is disrupted. “A. terreus having a disruption in the LDKSgene” comprises an A. terreus having a LDKS gene that is silenced bymethods known in the art. “A. terreus having a disruption in the LDKSgene” refers to an A. terreus that cannot produce functional LDKS. “A.terreus having a disruption in the LDKS gene” can also refer to an A.terreus that produces functional LDKS. The LDKS can be inactivated orinhibited by methods known in the art such as gene knock out protocols.The amount of LDKS present can be reduced by methods known in the art.Other methods of inhibition, inactivation, or disruption of LDKS gene orprotein include, but or not limited to, antisense, siRNA, RNAi, or RNAinterference as is known in the art. “LDKS gene” as used herein can alsorefer to the LovF gene. Disruption of the LovF gene is known in the art(see, e.g., U.S. Pat. No. 6,391,583 the contents of which are hereinincorporated by reference. “A. terreus having a disruption in the LDKSgene” is typically a genetically manipulated organism. Geneticmanipulation of A. terreus is known in the art. Gene disruption of theLov genes in A. terreus has been done previously (see, e.g., U.S. Pat.Nos. 6,391,583 and 6,943,017, the contents of which are hereinincorporated by reference). Disruption of specifically the LovF gene(producing LDKS) in A. terreus has been done previously (see, e.g., U.S.Pat. No. 6,943,017, the contents of which are herein incorporated byreference). Disruption of the LovF gene can occur by other methods as isknown in the art. A. terreus having a disruption in the LDKS gene can bein a fermentation mixture. Substrates can be added to the fermentationmixture of an A. terreus having a disruption in the LDKS gene to producelovastatin analogs.

“A component or method to increase the production of simvastatin” asused herein refers to a compound or substrate, synthetic or natural,that increases the production of certain intermediaries to increase theamount of simvastatin produced for scale-up and large-scale synthesis ofsimvastatin. Components and methods for increasing the production ofcertain intermediaries are known in the art. For example, compounds thatare added to the fermentation mixture to increase the amount ofintermediaries, such as monacolin J, in the production of lovastatin areknown in the art (see, e.g., U.S. Pat. No. 6,943,017, the contents ofwhich are herein incorporated by reference). Some of theseintermediaries, such as monacolin J, can also be used in the productionof simvastatin. Compounds for increasing the production of monacolin Jthereby can be added to increase the production of simvastatin. Forexample, compounds for increasing the production of monacolin J can bedirectly added to the fermentation mixture to increase the amount ofsimvastatin produced. An example of a component for increasing theproduction of simvastatin is a clone containing the D4B segment of thelovastatin producing gene cluster that is deposited in ATCC accessionnumber 98876. This clone can be transformed into a non-lovastatinproducing organism to produce monacolin J as is known in the art. Thisclone can also be transformed into a lovastatin-producing organism toincrease the production of monacolin J and thereby increase theproduction of simvastatin. Moreover, another example of a component forincreasing the production of simvastatin is the LovE/zinc finger gene,which can be transformed into a lovastatin-producing organism toincrease the production of simvastatin. Preferably, thislovastatin-producing organism would have a disruption in the LDKS gene(see, e.g., U.S. Pat. No. 6,391,583, the contents of which are hereinincorporated by reference). Components and methods to increase theproduction of simvastatin can refer to many others and are not limitedto the examples listed above.

As disclosed herein, an ““Acyl donor” or “acyl carrier” is a compoundhaving an acyl group that can be transferred to simvastatin and/or asimvastatin precursor or a related compound. Typically, ““Acyl donor” or“acyl carrier” is an acyl thioester that donates an acyl moiety to theC8 hydroxyl group of monacolin J. A wide variety of such agents areknown in the art that are further shown herein to have this activity(see, e.g. the illustrative acyl-thioesters in Table 1). In addition tothose known in the art and further shown by the instant disclosure tohave this activity, any potential acyl donor/carrier known in the art(or synthesized de novo) having an ability to acylate C8 of monacolin Jso as to produce simvastatin can be easily identified by comparativeexperiments with the acyl donors disclosed herein (e.g. acyl-SNAC). Asis known in the art, an acyl group can have the formula RCON; wherein Rcan be an alkyl or aryl and N can be —Cl, —OOCR, —NH2, —OR, or the like.Compounds that have an acyl group includes, but is not limited to, acidchlorides, esters, amides, or anhydrides and the like. These compoundscan be aliphatic or aromatic, substituted or unsubstituted. Examplesinclude, but are not limited to, benzoyl chloride, benzoic anhydride,benzamide, or ethyl benzoate, and the like. Other examples of acyldonors include, but are not limited to, α-dimethylbutyrl-SNAC,acyl-thioesters, acyl-CoA, butyryl-CoA, benzoyl-CoA, acetoacetyl-CoA,β-hydroxylbutyryl-CoA, malonyl-CoA, palmitoyal-CoA, butyryl-thioesters,N-acetylcyteamine thioesters (SNAC), methyl-thioglycolate (SMTG),benzoyl-SNAC, benzoyl-SMTG, or α-S-methylbutyryl-SNAC. These compoundscan be produced naturally or synthetically, and, in some cases, canpenetrate the cell membrane. A number of these compounds can be added toLovD in the presence of monacolin J to produce simvastatin for example.

“Acyl-SNAC” as used herein refers to α-dimethylbutyrl-SNAC. As is knownin the art, acyl-SNAC can penetrate the cell membrane under in vivoconditions. LovD can use acyl-SNAC as a substrate to initiate thereaction from monacolin J to simvastatin by regiospecifically acylatingthe C8 hydroxyl group of monacolin J. Acyl-SNAC can donate its acylgroup to LovD.

Typical Embodiments of the Invention

Those of skill in the art will understand that the disclosure providedherein allows artisans to produce a wide variety of embodiments of theinvention. A typical embodiment of the invention is a method of makingsimvastatin by combining together monacolin J; an acyl thioester thatdonates an acyl moiety to the C8 hydroxyl group of monacolin J in thepresence of LovD acyltransferase; and LovD acyltransferase; and thenallowing the LovD acyltransferase use an acyl group from the acylthioester to regioselectively acylate the C8 hydroxyl group of monacolinJ; so that simvastatin is made. In illustrative embodiments of theinvention, the LovD acyltransferase has the amino acid sequence of SEQID NO: 1 or SEQ ID NO: 2. A related embodiment of the invention is amethod of making simvastatin comprising the steps of combining togetherlovastatin; an acyl thioester that donates an acyl moiety to the C8hydroxyl group of monacolin J in the presence of LovD acyltransferase;and LovD acyltransferase. In this related method, the LovDacyltransferase is then allowed to hydrolyze lovastatin into monacolinJ; and to then use an acyl group from the acyl thioester toregioselectively acylate the C8 hydroxyl group of monacolin J; so thatsimvastatin is made. In certain embodiments, the simvastatin is made invitro in the absence of an isolated organism.

In some embodiments of the invention, the monacolin J; the acylthioester and the LovD acyltransferase are combined in a fermentationmedia in the presence of an isolated organism that produces the LovDacyltransferase and further wherein the organism is Escherichia coli,Aspergillus terreus, Monascus ruber, Monascus purpureus, Monascuspilosus, Monascus vitreus, Monascus pubigerus, Candida cariosilognicola,Aspergillus ogea, Doratomyces stemonitis, Paecilomyces virioti,Penicillum citrinum, Penicillin chgsogenum, Scopulariopsis brevicaulisor Trichoderma viride. Optionally, the isolated organism is Aspergillusterreus that expresses LovD polypeptide of SEQ ID NO: 1. In certainembodiments, the Aspergillus terreus does not express LovF polypeptideof SEQ ID NO: 3. In certain embodiments of the invention, the expressionpolypeptides such as those in SEQ ID NOs: 3 and 4 is reduced to at least90, 95, or 99% of is endogenous activity. Alternatively, the organismcan be Escherichia coli that expresses LovD polypeptide of SEQ ID NO: 1.In certain embodiments, the Escherichia coli does not express bioHpolypeptide of SEQ ID NO: 4. Consequently, the host can either be onethat produces a LovD acyltransferase endogenously or alternatively aheterologous host where the LovD acyltransferase gene has beenintroduced into the organism, for example, by a cloning technology knownin the art and further discussed herein. In a typical embodiment, theheterologous host that expresses LovD acyltransferase is a bacterium,yeast, or fungi that is known in the art to be useful for such purposes.

As discussed in detail below, the isolated organism can be grown underone of a variety of fermentation conditions known in the art and theexact conditions are selected, for example based upon fermentationparameters associated with optimized growth of a specific organism usedin an embodiment of the invention (see, e.g. Miyake et al., Biosci.Biotechnol. Biochem., 70(5): 1154-1159 (2006) and Hajjaj et al., Appliedand Environmental Microbiology, 67: 2596-2602 (2001), the contents ofwhich are incorporated by reference). Typically, the organism is grownat a temperature between 30-40° C., for a time period between at least 4to at least 48 hours. Typically, the organisms are grown at a pH between6.5-8.5. In certain embodiments of the invention, the pH of thefermentation media can be 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7,7.8, 7.9, 8.0 or 8.1. In illustrative embodiments, the organism is grownin a fermentation media comprising LB, F1 or TB media.

Optionally, the monacolin J that is combined with the other constituentsin the methods of the invention is produced by an isolated organismwithin the fermentation media, for example, one of the organisms listedabove that also produces the LovD acyltransferase. Alternatively, themonacolin J that is combined with the other constituents in the methodsof the invention is produced by a different organism that produces thiscompound that is added to the fermentation media and grows along withthe organism that produces the LovD acyltransferase. In anotherembodiment of the invention, monacolin J is derived from an exogenoussource and added to the fermentation mixture. Optionally, the method ofthe invention produces a composition of matter comprising 0%-1% of themonacolin J that was initially added to the combination. In certainembodiments of the invention, the method results in at least 95% of themonacolin J added to the combination being converted to simvastatin.

In typical embodiments of the invention, acyl thioester that can donatean acyl moiety to the C8 hydroxyl group of monacolin J in the presenceof LovD acyltransferase is derived from an exogenous source (e.g. achemical synthesis process) and added to the fermentation mixture. Avariety of such acyl thioesters are disclosed herein. Typically, theacyl thioester is a butyrlyl-thioester, a N-acetylcysteamine thioesteror a methyl-thioglycolate thioester. Optionally, the acyl thioestercomprises medium chain length (C3-C6) acyl group moieties. In certainembodiments of the invention, the acyl thioester is able to cross thecellular membranes of Escherichia coli or Aspergillus terreus cellsgrowing within a fermentation media. Typically, the acyl thioester isselected from the group consisting ofα-dimethylbutyryl-S-methyl-mercaptopropionate (DMB-S-MMP),dimethylbutyryl-S-ethyl mercaptopropionate (DMB-S-EMP) anddimethylbutyryl-S-methyl thioglycolate (DMB-S-MTG) anddimethylbutyryl-S-methyl mercaptobutyrate (DMB-S-MMB). In anillustrative embodiment, the acyl thioester isα-dimethylbutyryl-S-methyl-mercaptopropionate that is combined infermentation media in a concentration range of 1 mM-100 mM.

Certain embodiments of the methods for making simvastatin includefurther steps to purify simvastatin by the combination. For example,some embodiments of the invention include at least one purification stepcomprising lysis of cells of an isolated organism present in thecombination. Embodiments can include at least one purification stepcomprising centrifugation of cells or cell lysates of an isolatedorganism present in the combination. Embodiments can include at leastone purification step comprising precipitation of one or more compoundspresent in the combination. Embodiments can include at least onepurification step comprising filtration of one or more compounds presentin the combination. Embodiments can include at least one purificationstep comprising a high performance liquid chromatography (HPLC) analysisof one or more compounds present in the combination.

The disclosure provided herein shows that variety of permutations ofthese methods can be used to make simvastatin and huvastatin and thelike. In certain embodiments of the invention for example, the hostorganism produces the acyl thioester and/or the monacolin J.Alternatively, the acyl thioester and/or the monacolin J are added tothe organism as part of the process for producing simvastatin. Themethods can further comprise adding an expression vector having one ormore A. terreus genes that are known to facilitate the production ofsimvastatin and/or huvastatin or the like such as the genes that encodeSEQ ID NO: 1 or SEQ ID NO: 2 and transforming it into the heterologoushost, wherein the polypeptide having the acyltransferase activity isthereby expressed. In another embodiment, monacolin J can be producedfrom a heterologous host.

Another embodiment of the invention is a method of producing simvastatinfrom monacolin J in an organism which expresses a LovD acyltransferasegene comprising coculturing this first organism that expresses the LovDacyltransferase with a second organism (e.g. in a fermentation mixture)that produces the acyl thioester and/or the monacolin J, wherein theacyl thioester interacts with LovD acyltransferase gene product in thepresence of monacolin J to produce simvastatin. Optionally, the firstorganism is an organism that does not produce lovastatin naturally (e.g.E. coli transduced with the LovD acyltransferase gene). Alternatively,the first organism is a lovastatin-producing organism such as A. terreus(e.g. A. terreus having an inactivated LovF/LDKS gene). The method canfurther comprise adding one or more exogenous components to thefermentation mixture to increase the production of simvastatinprecursors such as monacolin J to thereby increase the production ofsimvastatin.

The methods of the invention can further comprise adding furthercomponents to the fermentation mixture to increase the production ofmonacolin J and to thereby increase the production of simvastatin and/orhuvastatin. As one illustrative embodiment of this method, the componentcan be a clone with the LovE gene, wherein the organism is transformedwith the clone and LovE is translated and thereby the production ofsimvastatin is increased. As another illustrative embodiment of thismethod, the component can be a clone containing the D4B segment of theA. terreus genome (ATCC accession 98876), wherein the organism istransformed with the clone so that the production of monacolin J isincreased.

Yet another embodiment of the invention is a method of convertingmonacolin J to simvastatin in vitro or in vivo in the presence of anexogenous acyl thioester. Preferably, the acyl thioester is capable ofpenetrating the cell membrane. In yet another embodiment of theinvention, is a method of converting monacolin J to simvastatin directlywithin the organism in the presence of the acyl thioester wherein theorganism produces LovD. In an illustrative embodiment of the invention,the organism is an A. terreus having a disrupted LDKS gene.

Yet another embodiment of the invention is a simvastatin product made bya process comprising the steps of combining together monacolin J; anacyl thioester that donates an acyl moiety to the C8 hydroxyl group ofmonacolin J in the presence of LovD acyltransferase; and LovDacyltransferase; and allowing the LovD acyltransferase use an acyl groupfrom the acyl thioester to regioselectively acylate the C8 hydroxylgroup of monacolin J so that the simvastatin product is made. A relatedembodiment of the invention is a huvastatin product made by a processcomprising the steps of combining together hydrolyzed pravastatintetra-ol; an acyl thioester that donates an acyl moiety to the C8hydroxyl group of hydrolyzed pravastatin tetra-ol in the presence ofLovD acyltransferase; and LovD acyltransferase; and allowing the LovDacyltransferase use an acyl group from the acyl thioester toregioselectively acylate the C8 hydroxyl group of hydrolyzed pravastatintetra-ol; so that the huvastatin product is made.

Another embodiment of the invention is a method of producing simvastatincomprising hydrolyzing lovastatin into monacolin J in the presence ofLovD of SEQ ID NO: 1 or a LovD homologue and acylating monacolin J,wherein said hydrolyzation and acylation produce simvastatin. Anotherrelated embodiment of the invention is a method of producing huvastatincomprising pravastatin into hydrolyzed pravastatin in the presence ofLovD of SEQ ID NO: 1 or a LovD homologue and acylating a monacolin Jvariant, wherein said hydrolyzation and acylation produce huvastatin.

Embodiments of the invention include composition of matter used to makeand or made by the processes disclosed herein. For example, oneembodiment of the invention is a composition of matter comprisingmonacolin J; an acyl thioester that donates an acyl moiety to the C8hydroxyl group of monacolin J in the presence LovD acyltransferase.Certain embodiments of these compositions of matter further compriseLovD acyltransferase. Certain embodiments of these compositions ofmatter further comprise simvastatin. Optionally, the composition furthercomprises an isolated organism such as Escherichia Aspergillus terreus,Monascus ruber, Monascus purpureus, Monascus pilosus, Monascus vitreus,Monascus pubigerus, Candida cariosilognicola, Aspergillus ogea,Doratomyces stemonitis, Paecilomyces virioti, Penicillum citrinum,Penicillin chysogenum, Scopulariopsis brevicaulis or Trichoderma viride.In typical embodiments, the organism in the composition is Aspergillusterreus or Escherichia coli that expresses LovD polypeptide of SEQ IDNO:1. In one embodiment of the invention the organism is Aspergillusterreus that does not express LovF polypeptide of SEQ ID NO: 3. Inanother embodiment of the invention the organism is Escherichia colithat does not express bioH polypeptide of SEQ ID NO: 4. In certainembodiments of the invention, isolated organism within the compositionhas been transduced with an expression vector encoding Aspergillusterreus LovD polypeptide of SEQ ID NO: 1.

A variety of acyl thioesters that can be used in the compositions of theinvention are disclosed herein. Typically, the acyl thioester is abutyrlyl-thioester, a N-acetylcysteamine thioester or amethyl-thioglycolate thioester. Optionally, the acyl thioester comprisesmedium chain length (C3-C6) acyl group moieties. In certain embodimentsof the invention, the acyl thioester is able to cross the cellularmembranes of Escherichia coli or Aspergillus terreus cells growingwithin a fermentation media. Typically, the acyl thioester is selectedfrom the group consisting ofα-dimethylbutyryl-S-methyl-mercaptopropionate (DMB-S-MMP),dimethylbutyryl-S-ethyl mercaptopropionate (DMB-S-EMP) anddimethylbutyryl-S-methyl thioglycolate (DMB-S-MTG) anddimethylbutyryl-S-methyl mercaptobutyrate (DMB-S-MMB). In anillustrative embodiment, the acyl thioester isα-dimethylbutyryl-S-methyl-mercaptopropionate that is combined infermentation media in a concentration range of 1 mM-100 mM and cantypically be about 10, 20, 30, 40, 50, 60, 70, 80 or 90 mM. In someembodiments of the invention, the composition further compriseslovastatin and the amount of simvastatin in the composition is greaterthan the amount of lovastatin in the composition.

As is discussed in detail below, the methods and materials of theinvention that are used to make simvastatin can be adapted to producecompounds that are structurally similar to simvastatin, for examplehuvastatin. In this context, one embodiment of the invention is a methodof making huvastatin comprising the steps of combining togetherhydrolyzed pravastatin tetra-ol; an acyl thioester that donates an acylmoiety to the C8 hydroxyl group of hydrolyzed pravastatin tetra-ol inthe presence of LovD acyltransferase; and LovD acyltransferase; and thenallowing the LovD acyltransferase use an acyl group from the acylthioester to regioselectively acylate the C8 hydroxyl group ofhydrolyzed pravastatin tetra-ol, so that huvastatin is made. A relatedembodiment of the invention is a composition of matter comprising:hydrolyzed pravastatin tetra-ol; an acyl thioester that donates an acylmoiety to the C8 hydroxyl group of hydrolyzed pravastatin tetra-ol inthe presence LovD acyltransferase; LovD acyltransferase; and huvastatin.

Yet another embodiment of the invention is a composition of mattercomprising one or more of the huvastatin precursors, for example,hydrolyzed pravastatin tetra-ol or 6-hydroxyl-6-desmethylmonacolin J inthe presence of thioester selected for its ability to acylate the C8hydroxyl group of monacolin J or a monacolin J variant such ashydrolyzed pravastatin tetra-ol. Typically, such compositions canfurther include an organism as discussed above. Consequently,embodiments of the invention include processes for making simvastatin orhuvastatin composition of matter substantially as herein disclosed andexemplified.

In situations where a modified organism that produces simvastatin (e.g.A. terreus with a disruption in the LDKS gene) also produces lovastatin(e.g. some minimal residual amount), the methods and materials disclosedherein allow the manipulation of the biochemical pathways/processes inthe organism so that simvastatin or a related compound such ashuvastatin is the predominant product of these pathways. A relatedembodiment is a composition of matter comprising an organism andsimvastatin and/or huvastatin produced by that organism, wherein theamount of simvastatin and/or huvastatin in the composition is greaterthan the amount of lovastatin in the composition.

A related embodiment of the invention is a composition of mattercomprising LovD, hydrolyzed pravastatin, and an acyl thioester. Inillustrative embodiments, the composition of matter comprises an E. colithat produces huvastatin. In related embodiments, the composition ofmatter is an A. terreus (e.g. A. terreus with a disruption in the LDKSgene) that produces huvastatin. In situations where a modified organismthat produces huvastatin (e.g. A. terreus with a disruption in the LDKSgene) also produces lovastatin (e.g. some minimal residual amount), themethods and materials disclosed herein allow the manipulation of thebiochemical pathways/processes in the organism so that huvastatin is thepredominant product of these pathways. A related embodiment is acomposition of matter comprising an organism and huvastatin produced bythat organism, wherein the amount of huvastatin in the composition isgreater than the amount of lovastatin in the composition.

In certain embodiments of the invention, further components and/ormethodological steps can be combined with one or more of the methods andmaterials discussed above. For example, the methods can further compriseusing high cell-density fermentation to increase the effectiveconcentration of LovD acyltransferase and optimise fermentationconditions and/or increasing LovD acyltransferase catalytic efficienciestowards the one or more acyl thioesters via protein engineering. Manyother components or methods can be used to increase the production ofsimvastatin or of an intermediary compound that facilitates theproduction of simvastatin.

As noted above, lovastatin production (which requires LovDacyltransferase activity) is observed in a number of organisms such asAspergillus terreus, Monascus ruber, Monascus purpureus, Monascuspilosus, Monascus vitreus, Monascus pubigerus, Candida cariosilognicola,Aspergillus ogea, Doratomyces stemonitis, Paecilomyces virion,Penicillum citrinum, Penicillin chgsogenum, Scopulariopsis brevicaulisor Trichoderma viride. In view of the nature of the invention disclosedherein, using known methods and materials in combination with thedisclosure provided herein, one of skill in the art can combine aculture of one or more of these organisms (or any other organism knownto produce lovastatin) with monacolin J and acyl thioester that donatesan acyl moiety to the C8 hydroxyl group of monacolin J in the presenceLovD acyltransferase (e.g. α-dimethylbutyrl-SNAC) to use LovDacyltransferase to make simvastatin. As shown by the instant disclosurefor example, the nature of the instant invention allows one to makesimvastatin and related compounds in the wide variety of lovastatinproducing organism known in the art, without any specific knowledgeregarding the characteristics of the specific LovD acyltransferaseexpressed in these organisms.

In an illustrative procedure for making simvastatin in a new organism(e.g. a fungal species related to A. terreus) and/or testing theorganism for its ability to do so, a first step is to make a mixture ofmonacolin J and acyl thioester that donates an acyl moiety to the C8hydroxyl group of monacolin J. In a second step, this mixture is thencombined with the organism (e.g. a lovastatin producing organism) in afermentation mixture and allowed to grow. In a third step, the mixtureis then tested for the presence of simvastatin, for example, by using aHPLC analysis as are discussed in the examples below. In view of thehigh throughput screening methodologies known in this art (see, e.g.Kittel et al., Metab. Eng. 2005, 7(1): 53-58, which is incorporatedherein by reference), such methods can be performed on a large number oftest samples such as the huge number of fungal cultures that are knownin the art and readily available to the artisan so as to easilydetermine where and which species of organisms express polypeptides thatpossess a LovD acyltransferase activity that allows them to be used inthe embodiments of the invention disclosed herein.

The methods disclosed herein also allow artisans to isolate and clonefurther LovD acyltransferase embodiments, for example, those embodimentswhich may have additional desirable qualities such as enhanced stabilityunder various reaction conditions and/or favorable enzymatic kineticsunder various reaction conditions. In this context, another embodimentof the invention is a method for identifying further LovDacyltransferase embodiments of the invention, the method comprising thesimple steps of combining an organism likely to express LovDacyltransferase (e.g. a lovastatin producing organism) or a cell extractfrom that organism with an acyl thioester that donates an acyl moiety tothe C8 hydroxyl group of monacolin J in the presence LovDacyltransferase (e.g. α-dimethylbutyrl-SNAC) and then testing thiscombination for the presence of simvastatin. The presence of simvastatinis indicative of the presence of the desired LovD acyltransferaseactivity. In view of the methods disclosed herein as well as highthroughput screening methodologies known in this art (see, e.g. Kittelet al., Metab. Eng. 2005, 7(1): 53-58, which is incorporated herein byreference), such methods can be performed on a large number of testsamples such as the huge number of fungal cultures that are known in theart and readily available to the artisan (e.g. lovastatin producingcultures) with only a minimal amount of experimentation. Consequently,the large number of lovastatin producing organisms known in the art, thehigh throughput screening methodologies known in this art and theinstant disclosure guide a worker to easily determine where and whichspecies of polypeptides possess a LovD acyltransferase activity thatallows them to be used in the embodiments of the invention disclosedherein.

Once LovD acyltransferase activity is observed using these methods, itcan then be used in art accepted methods to clone the gene encoding theprotein having this activity. Alternatively, a library of an organism'sgenes can then be screened using a LovD acyltransferase polynucleotidesequence (e.g. from A. terreus) either alone or in combination withfunctional studies as discussed above in order to identify a new a LovDacyltransferase polynucleotide sequence having homology to SEQ ID NO: 1or SEQ ID NO: 2). In this context, homologous enzymes to LovD that canbe found in, for example, but not limited to, fungal polyketide geneclusters. For example, Mlc in the compactin biosynthetic pathway isimplicated to catalyze the identical transacylation reaction (see, e.g.,Y. Abe, T. et. al., Mol Genet Genomics. 2002, 267, 636-646), whereas anacyltransferase in the squalestatin pathway can catalyze a similarreaction between an ACP-bound tetraketide thioester and an aglycon (see,e.g., R. J. Cox, F. et. al., Chem Commun (Camb) 2004, 20, 2260-2261).The amino acid sequence of LovD resembles type C β-lactamase enzymes,which catalyze the hydrolytic inactivation of the β-lactam class ofantibiotics (see, e.g., E. Lobkovsky, E. M. et. al., Biochemistry, 1994,33, 6762-6772 and A. Dubus, D. et. al., Biochem. J. 1993, 292, 537-543).Alignment of LovD with the Enterobacter cloacae P99 lactamse (see, e.g.,S. D. Goldberg, et. al., Protein Sci. 2003, 12, 1633-1645) showsmoderate sequence homology, including potentially conserved active siteresidues, such as the catalytic Ser76, Lys79, Tyr188, and Lys315 (see.e.g. S. D. Goldberg, et. al., Protein Sci. 2003, 12, 1633-1645). In viewof these methods, yet another embodiment of the invention is an isolatedLovD acyltransferase polypeptide having at least 80%, 85%, 90%, 95%,96%, 97%, 98%, or 99% sequence identity with the amino acid sequence setout in SEQ ID NO:1, wherein the polypeptide has the ability to acylatethe C8 hydroxyl group of monacolin J in the presence of an appropriateacyl thioester donor (e.g. α-dimethylbutyrl-SNAC).

As disclosed herein, an “acyl thioester to regioselectively acylate theC8 hydroxyl group of monacolin J” is a compound having an acyl groupthat can be transferred to monacolin J or a related compound so as tomake simvastatin or a related compound as disclosed herein. A widevariety of such agents are known in the art that are further shownherein to have this activity (see, e.g. the illustrative acyl-thioestersin Table 1). In addition to those known in the art and further shown bythe instant disclosure to have this activity, any potential acyldonor/carrier known in the art (or synthesized de novo) that further hasan ability to acylate C8 of monacolin J so as to produce simvastatin canbe easily identified by comparative experiments with the acyl donorsdisclosed herein (e.g. acyl-SNAC). Typically in such experiments, theacyl thioester is a butyrlyl-thioester, a N-acetylcysteamine thioesteror a methyl-thioglycolate thioester. Optionally, the acyl thioestercomprises medium chain length (C3-C6) acyl group moieties. In certainembodiments of the invention, the acyl thioester is able to cross thecellular membranes of Escherichia coli or Aspergillus terreus cellsgrowing within a fermentation media. Typically, the acyl thioester isselected from the group consisting ofα-dimethylbutyryl-S-methyl-mercaptopropionate (DMB-S-MMP),dimethylbutyryl-S-ethyl mercaptopropionate (DMB-S-EMP) anddimethylbutyryl-S-methyl thioglycolate (DMB-S-MTG) anddimethylbutyryl-S-methyl mercaptobutyrate (DMB-S-MMB).

As shown by the instant disclosure, the nature of the instant inventionallows one to readily identify a compound as a “acyl thioester toregioselectively acylate the C8 hydroxyl group of monacolin J” withminimal experimentation. In one illustrative procedure for identifying acompound as a “acyl thioester to regioselectively acylate the C8hydroxyl group of monacolin J”, a first step is to make a mixture ofmonacolin J and A. terreus. In a second step, this mixture is thencombined with a test compound in a fermentation mixture and allowed togrow. In a third step, the mixture is then tested for the presence ofsimvastatin, for example by using a HPLC analysis as are discussed inthe examples below, wherein the presence of simvastatin identifies thecompound as having this activity. In view of the high throughputscreening methodologies known in this art (see, e.g. Kittel et al.,Metab. Eng. 2005, 7(1): 53-58, which is incorporated herein byreference), such methods can be performed on a large number of testsamples so as to easily determine where and which species of acyl donorcompounds possess a utility that allows them to be used in theembodiments of the invention disclosed herein.

Embodiments of the invention also include articles of manufacture and/orkits designed to facilitate the methods of the invention. Typically suchkits include instructions for using the elements therein according tothe methods of the present invention. Such kits can comprise a carriermeans being compartmentalized to receive in close confinement one ormore container means such as vials, tubes, and the like, each of thecontainer means comprising one of the separate elements to be used inthe method. For example, one of the containers can comprise a vial, forexample, containing A. terreus having a disruption in the LDKS gene andanother vial containing an acyl-SNAC compound or the like, both of whichcan be added to a fermentation mixture to produce simvastatin.

In a typical embodiment of the invention, an article of manufacturecontaining materials useful for production of simvastatin is provided.The article of manufacture comprises a container and a label. Suitablecontainers include, for example, bottles, vials, syringes, and testtubes. The containers may be formed from a variety of materials such asglass or plastic. The container can hold a composition of matter (e.g.an acyl carrier and an organism) which can produce simvastatin, forexample. The label on, or associated with, the container indicates thatthe composition is used for examining cellular polypeptides. The articleof manufacture may further comprise a second container comprisinganother compound or substrate for addition to the fermentation mixturefor example. This compound or substrate, for example, might be used toincrease the production of certain intermediaries in the production ofsimvastatin, such as monacolin J. It may further include other materialsdesirable from a commercial and user standpoint, including otherbuffers, diluents, filters, needles, syringes, and package inserts withinstructions for use.

Further biological aspects of the invention are discussed in thefollowing sections.

Biochemical Aspects of Embodiments of the Invention

An appreciation of certain aspects of the invention is facilitated bydiscussions of biochemical aspects of the invention. In the followingsections and examples of the specification, the LovD acyltransferase isthe A. terreus LovD polypeptide shown in SEQ ID NO: 1, and is typicallyreferred to as “LovD” or the “LovD enzyme”.

The promiscuity of the LovD enzyme towards alternative acyl donors wasexamined. We found that the acyl carrier/donor does not need to beattached to the LDKS. Alternative thiol containing carriers aresuitable, including acyl-CoA (coenzyme A) and more importantly, themembrane permeable acyl-SNAC (FIG. 7). We found that LovD does not needto interact with LovF and can accept an acyl group from a variety ofdifferent donors. We also found that LovD can transfer a variety of acylsubstrates to the C8-hydroxyl group of monacolin J to yield anassortment of lovastatin analogs. We found that LovD canregioselectively acylate the C8 hydroxyl position in monacolin J with avariety of acyl substrates. Amongst the successful acyl groups isα-dimethylbutyryl-SNAC, which yields simvastatin in the presence of LovDand monacolin J. We also found that the transacylation activity of LovDbe confirmed in vitro and be reconstituted in a heterologous host. Wefound that LovD directly acylates monacolin J with α-dimethybutyrate toyield the pharmaceutically important simvastatin. α-Dimethylbutyryl-SNACis cell-permeable. The cell permeable properties ofα-dimethylbutyryl-SNAC has an important implication: the compound can besupplied as a precursor in vivo to an organism, such as a prokaryote oran eukaryote, expressing LovD. The prokaryote or eukaryote, whenfermented in the presence of monacolin J (either made endogenously, orsupplied exogenously to the fermentation media) can directly affordsimvastatin. E. coli, for example, was examined as a microbial host forthe bioconversion of monacolin J into simvastatin. Both monacolin J anddimethylbutyryl-SNAC were added to a growing culture of an E. colistrain overexpressing the LovD enzyme (FIG. 10). Simvastatin wasisolated from the fermentation broth of the culture in good yield. Thistechnique can be used in the native lovastatin producer Aspergillusterreus. A strain that is deficient in LDKS can be constructed so thatit will not be able to synthesize the 2-methylbutyrate side chain.α-Dimethylbutyryl-SNAC can be added to the fermentation medium.Simvastatin can be synthesized during this single step fermentation.

As noted above, LovD can catalyze the final acyl transfer step duringlovastatin biosynthesis and can regiospecifically acylate the C8hydroxyl group in monacolin J. LovD can display broad substratespecificity towards the decalin aglycon, the acyl carrier, and the acylgroup. When supplemented with the unnatural substrateα-dimethylbutyryl-SNAC, LovD can produce the pharmaceutically importantsimvastatin both in vitro and in vivo. When anα-dimethylbutyryl-thioester precursor is supplied to a LovF-deficientstrain of A. terreus, the lovastatin biosynthetic pathway can beredirected to afford simvastatin directly. This invention allows for: 1)Microbial conversion of monacolin J to simvastatin (FIG. 10); 2)Coculturing of the LovD overexpression strain with a strain thatproduces monacolin J; and 3) Single step fermentation of Aspergillusterreus (ΔLDKS) to yield simvastatin. In each case, the acyl substrateα-dimethylbutyryl-SNAC can be synthesized chemically and be added to thefermentation broth.

LovD does not Need to Interact with LovF, and LovD can Catalyze theTransacylation Reaction of Acyl-CoA and Other Alternate Thiol-ContainingCarriers (FIG. 5)

We first examined if an acyl-CoA can be used as substrate for thetransacylation reaction. The uninterrupted gene of LovD was amplifiedfrom A. terreus genomic DNA using splice by overlap extension PCR andwas inserted into the pET28a expression vector. Overexpression ofsoluble, N-terminal 6×His fusion LovD was performed in E. coli strainBL21 (DE3)/pAW31. LovD was purified using Nickel-NTA affinity column tohomogeneity (final yield ˜40 mg/L). The substrate monacolin J (1 mM) wasprepared through the LiOH hydrolysis of lovastatin and was added to areaction mixture containing pure LovD (10 μM) and butyryl-CoA (4 mM),which is a commercially available acyl-CoA that best mimics the naturalα-methylbutyrate side chain. The reaction mixture was incubated at roomtemperature, extracted with ethyl acetate and analyzed by HPLC and LC-MS(FIG. 5).

A single, more hydrophobic compound with an identical UV absorbance aslovastatin was formed in the reaction mixture, in conjunction with thedisappearance of monacolin J (FIG. 5, trace a). The mass of the newcompound was found to be 391 (M+H), in accordance with addition of abutyryl group to monacolin J. The selective esterification of C8hydroxyl group of monacolin J to yield 4 is confirmed by proton NMRspectroscopy (CDCl₃, 500 MHz). The proton NMR of 4 (lactonized form) isnearly identical to that of lovastatin except the aliphatic signals ofthe linear acyl side chain. The diagnostic H8 multiplet (assigned using¹H-¹H COSY, ¹H-¹³C HMQC and ¹H-¹³C HMBC) in 4 is shifted downfield to δ5.38, compared to δ 4.23 observed for the same proton in monacolin J.This is consistent with the deshielding effect of the acyloxysubstitution at C8. Protons H11 and H13 of carbons bearing otherhydroxyl groups were shifted from δ 4.71 and δ 4.38 in monacolin J to δ4.51 and δ 4.36 in 4, respectively.

As shown in FIG. 5A (trace a), 87% conversion of monacolin J to 4 wasobserved after 10 hours using butyryl-CoA as an acyl donor. Atime-course study revealed an apparent turnover rate of 0.18 min⁻¹ (FIG.5B). The observed 87% conversion is likely approaching equilibrium asevident in FIG. 5B. To examine the biochemical nature of the equilibriumconversion, we assayed whether LovD also catalyzes the reverse,hydrolysis reaction. Indeed, when lovastatin was used as the onlysubstrate, formation of monacolin J was readily detected with k_(cat)and K_(m) values of 0.21±0.01 mind and 0.56±0.05 mM, respectively (FIG.5C). When the putative active site serine in LovD (Ser76) was mutated toan alanine, formation of 4 or the hydrolysis of lovastatin cannot bedetected (FIG. 5A, trace b).

The enzymatic synthesis of 4 confirms that LovD indeed catalyzes theacyl transfer reaction shown in FIG. 4. Furthermore, this result showthat direct association between domains of LovF and LovD is not requiredfor catalytic turnover, in contrast to the previously hypothesized modeof LovD catalysis (see, e.g., J. Kennedy, K. et. al., Science, 1999,284, 1368-1372 and C. R. Hutchinson, J. et. al., Antonie Van Leeuwenhoek2000, 78, 287-295). Acyl-S-CoA can substitute for acyl-S-LovF, albeitlikely with a significantly higher K_(m) due to the loss of potentialprotein-protein interactions.

Transacylation Assays with Several Different Commercially Available AcylSubstituents Revealed LovD's Preference Towards Medium Chain Length(C3-C6) Acyl Groups

We assayed the tolerance of LovD towards different acyl substituents byperforming the transacylation assay with various commercially availableacyl-CoAs (Table 1A). All assays were perform with 1 mM monacolin J, 4mM acyl-CoA and 10 uM LovD for 10 hours. Our results clearly indicateLovD displays preference towards medium chain length (C3-C6) acyl groupswith butyryl-CoA being the optimal alkylacyl-CoA substrate.Surprisingly, the bulkier benzoyl-CoA was one of the best acyl substrateexamined, with nearly 70% conversion of simvastatin to the corresponding8-benzoxy-lovastatin analog (apparent k_(cat)=0.16 mind). Introducingα-β unsaturation significantly decreased the reaction rate, as seen inthe 6% acylation of monacolin J in the presence of crotonyl-CoA.Acetoacetyl-CoA and β-hydroxylbutyryl-CoA were both excellent substratesof LovD, in good agreement with the isolation of monacolin X (see, e.g.,A. Endo, K. et. al., J. Antibiot, 1986, 38, 321-327) and monacolin M(see, e.g., A. Endo, D. et. al., J. Antibiot. 1986, 39, 1670-1673) fromthe natural host, respectively. Among the CoA substrates assayed, LovDwas inactive towards malonyl- and palmitoyl-CoA.

Further Transacylation Assays with an Alternative Acyl Carrier,Acyl-SNAC, which is Simpler to Prepare Synthetically and can PenetrateCell Membrane Under In Vivo Conditions, Revealed that Acyl-SNAC is aCompetent Substrate for LovD

To further examine the substrate specificities of LovD towardsalternative acyl carriers, especially those that are simpler to preparesynthetically, and can penetrate cell membrane under in vivo conditions,we assayed two variants of butyryl-thioesters as substrates of LovD(FIG. 5). N-acetylcysteamine thioesters (SNAC) have been usedextensively as probes and precursors in studying natural productbiochemistry (see, e.g. Auclair et al., Science, 1997, 277, 367-369).Methyl-thioglycolate (SMTG) was recently shown to be a cost-effectivesubstitute for SNAC in the precursor-directed biosynthesis oferythromycin (see, e.g., S. Murli, K. S. et. al., Appl. Environ.Microbiol. 2005, 71, 4503-4509). FIG. 5 (traces c and d) shows theconversion of monacolin J to 4 when these butyryl-thioesters were usedas acyl donors. Both SNAC and SMTG thioesters substituted forbutyryl-CoA efficiently, with apparent k_(cat) values of 0.09 min⁻¹ and0.23 min⁻¹ (FIG. 5B), respectively, further highlighting thatprotein-protein interactions between LovD and LovF, as well as theinteraction between LovD and the phosphopantetheine arm are not requiredfor acyl transfer. Butyryl-thioethane, however, was not a competentsubstrate of LovD and supported only 4% conversion of monacolin J to 4.Similarly, benzoyl-SNAC and benzoyl-SMTG substituted for benzyl-CoAefficiently, with apparent k_(cat) values of 0.12 min⁻¹ and 0.15 min⁻¹,respectively (Table 1A).

Assay for the In Vitro Chemoenzymatic Synthesis of Lovastatin andSimvastatin Using A-S-Methylbutyryl-SNAC and A-Dimethylbutyryl-SNACRevealed that Monacolin J is Converted to Simvastatin in the Presence ofAcyl-SNAC

We then synthesized α-S-methylbutyryl-SNAC and α-dimethylbutyryl-SNACand assayed for the in vitro chemoenzymatic synthesis of lovastatin andsimvastatin, respectively. The results are shown in FIG. 8 and Table 1A.Authentic samples of lovastatin and simvastatin were used as referencesfor HPLC detection (FIG. 8, trace a). The natural, α-S-methylbutyrateside chain was surprisingly a poorer substrate compared to butyryl-,pentanoyl- and hexanoyl-SNAC. The apparent k_(cat) (˜0.04 min⁻¹) oflovastatin synthesis is more than 50% slower than that of LovD towardsbutyryl-SNAC. This suggested that the wild type LovD has not beenoptimized towards transferring the branched substrate. Addition of asecond methyl substituent at the α-position further attenuated the rateof acylation, likely attributed to the increased steric hindrance of thedimethyl moiety. Under standard assay conditions, approximately 10% ofmonacolin J was converted to simvastatin when α-dimethylbutyryl-SNAC isused as a substrate (apparent k_(cat)=0.02 min⁻¹). We were able to reachequilibrium conversions >70% when 100 uM LovD and a 10 fold excess ofα-dimethyl-SNAC or α-dimethyl-SMTG were added to the in vitro reactionmixture (FIG. 8, traces b and c).

LovD, In Vitro, can Transfer a Variety of Acyl Substrates (not JustLDKS) to the C-8-Hydroxyl Group of Monacolin J to Yield an Assortment ofLovastatin Analogs

To test the substrate specificity of LovD towards monacolin J variants,we assayed the conversion of tetra-ol 7 to 8, pravastatin (5) andhuvastatin (6) (FIG. 9). It has been shown previously shown that when8-desmethyl-monacolin J is fed to A. terreus mutant blocked in monacolinJ biosynthesis, compactin can be readily isolated (see, e.g., J. L.Sorensen, K. et. al., Org. Biomol. Chem. 2003, 1, 50-59). This hintsthat LovD may be tolerant of substitutions at the C6 position of thedecalin core. Indeed, LovD displays relaxed specificity towards thehydroxyl substitution at C6 and catalyzed the acylation of 7 with higherefficiencies. The apparent turnover rates for the synthesis of 8,pravastatin, or huvastatin using the corresponding acyl-thioesters were0.18, 0.11, 0.03 min⁻¹, respectively. The retention time of authenticpravastatin was identical to the enzymatically synthesized compound. Themass of the newly formed compounds was verified by LC-MS. We did notdetect any di-acylated products in reaction mixture.

In Vivo Data Show that LovD can be Used for Preparative Biosynthesis ofLovastatin Analogs

To demonstrate LovD can be used for preparative biosynthesis oflovastatin analogs, we first attempted to perform the benzylationreaction in vivo using E. coli as a heterologous host. TheBL21(DE3)/pAW31 overexpression strain was grown in a shake flask (200mL) to an OD₆₀₀ of 1.0, at which time 1 mM IPTG, 0.8 mM monacolin J and4 mM of either benzoyl-SNAC or benzoyl-SMTG were added to the culture.Expression of LovD and bioconversion was performed at 18° C. The culturewas extracted and analyzed for the formation of 8-benzoyl-monacolin J.When supplemented with benzoyl-SNAC, 84% conversion of monacolin J wasdetected within 20 hours post-induction. The product was lactonized andpurified by a single silica-gel chromatography step. The NMR spectraconfirmed the regioselective benzoylation of the C8 hydroxyl group asthe diagnostic H8 multiplet is shifted downfield to δ 5.61. In contrast,only 40% benzoylation was observed for the culture that was supplementedwith benzoyl-SMTG. Benzoyl-SMTG was rapidly degraded by E. coli and notrace can be detected in the culture medium after 24 hours.

An Acyl Substrate, A-Dimethylbutyrl-SNAC Yielded Simvastatin in thePresence of Purified LovD and Monacolin J

We performed low cell-density fermentation with α-dimethylbutyryl-SNACas a precursor to yield simvastatin from monacolin J in a singlebiosynthetic step. After two days of culturing, we observed ˜90%conversion of monacolin J to simvastatin (see, e.g., “fermentationconditions” listed below). The product was purified and the proton andcarbon NMR spectra were identical to those of the commercially purchasedcompound. The lower yield of simvastatin is consistent with the slowerturnover rate observed in vitro, which may be further decreased at lowerintracellular concentration of the SNAC precursor. Other factors, suchas the reversible hydrolysis of simvastatin (we observed simvastatinhydrolysis in the presence of LovD—the k_(cat) (0.02 min⁻¹) ofhydrolysis is ˜10-fold slower than that of lovastatin hydrolysis shownin FIG. 5C) and inactivation of LovD after prolonged fermentation maylead to the observed conversion. We anticipate the yield can besignificantly improved by several means, such as 1) use highcell-density fermentation to increase the effective concentration ofLovD and optimize fermentation conditions; 2) increase LovD catalyticefficiencies towards the unnatural precursor by protein engineering.

Throughout this application, various publications are referenced. Thedisclosures of these publications are hereby incorporated by referenceherein in their entireties.

EXAMPLES

The Examples below provide illustrative methods and materials that canbe used in the practice the various embodiments of the inventiondisclosed herein.

Example 1: Cloning, Expression and Purification of Illustrative LovDAcyltransferase

The three exons of A. terreus LovD were individually amplified from thegenomic DNA of A. terreus and spliced to yield a continuous open readingframe using splice by overlap extension PCR. The restriction sites NdeIand HindIII were introduced on the 5′ and 3′ outside primers,respectively. The gene cassette was ligated into pET28 (Novagen) toyield the expression construct pAW31. The E. coli BL21(DE3) straintransformed with pAW31 was grown in LB medium at 37° C. to a OD₆₀₀ of0.5, at which time 1 mM IPTG was added to the culture and expression wasperformed at 18° C. for 24 hours. Cells were collected bycentrifugation, resuspended in Buffer A (50 mM Tris, pH. 8.0, 2 mM DTT,2 mM EDTA) and were lysed by sonication. Cell debris and insolubleproteins were removed by centrifugation (17,000 g, 4° C., 1 hour). Tothe cleared lysate, 2 mL of Ni-NTA resin (Qiagen) was added. LovD waspurified using a step gradient of buffer A with increasing concentrationof imidazole. Pure (>95%) LovD proteins were eluted at buffer Acontaining 250 mM imidazole, buffer exchanged into Buffer A withoutimidazole, concentrated, aliquoted and flash frozen. The frozen LovDaliquots were of single use only. We observed significant decrease inenzyme activity after repeated freeze-thaw cycles.

Example 2: Illustrative Fermentation Conditions

Fermentation conditions: 500 mL culture with LB media with 30 mg/Lkanamycin. At OD₆₀₀ of 1.0, cells were concentrated to a final OD₆₀₀ of5.0 and induced with 1 mM IPTG. Substrates monacolin J andα-dimethylbutyryl-SNAC were added to a final concentration of 1 mM and 4mM. At different time points, culture samples were collected,centrifuged, filtered and injected on to HPLC (20 μL). Extractionconditions: When maximum conversion was reached, the broth was acidifiedto pH of 2.0, extracted with ethyl acetate, dried and redissolved intoluene. The lactone form of monacolin J was obtained by refluxing usinga soxhlet apparatus as discussed before (see, e.g., J. L. Sorensen, K.et. al., Org. Biomol. Chem. 2003, 1, 50-59).

A variety of fermentation media such as LB, F1 or TB fermentation mediaare well known in the art which can be used or adapted for use withembodiments of the invention disclosed herein including LB, TB and F1media. Further media tailored to growing organisms such as A. Terreusand M. pilosus are also well known in the art (see, e.g. Miyake et al.,Biosci. Biotechnol. Biochem., 70(5): 1154-1159 (2006) and Hajjaj et al.,Applied and Environmental Microbiology, 67: 2596-2602 (2001), thecontents of which are incorporated by reference).

A typical Luria Bertani Broth (LB) Recipe is as follows:

-   -   10 g Tryptone    -   5 g Yeast Extract    -   10 g NaCl    -   1 L distilled water

pH to ˜7.3-7.5.

A typical TB Recipe is as follows:

-   -   3 g Pipes (10 mM)    -   2.2 g CaCl2 H₂O (15 mM)    -   18.6 KCl (250 mM)    -   10.9 g MnCl2 (55 mM)    -   1 L distilled water

pH to 6.7-6.8 with KOH

A typical F1 media can be found in U.S. Pat. No. 5,064,856, the contentsof which are incorporated herein by reference.

Typically such media is sterilized after preparation by procedures suchas filtration or autoclaving.

Example 3: Improving Simvastatin Bioconversion in Escherichia Coli byDeletion of BioH

This example further characterizes the LovD polypeptide encoded in thelovastatin gene cluster (see also Xie et al., (2006) Chem Biol 13:1161-1169). LovD catalyzes the last step of lovastatin biosynthesis andis responsible for transferring the 2-methylbutyrate side chain from themegasynthase LovF to the immediate biosynthetic precursor, monacolin J(MJ) acid (see, e.g., Kennedy et al., (1999) Science 284: 1368-1372). Wedemonstrated that LovD displays broad substrate specificity towards thedecalin core, the thioester acyl unit and the thioester acyl carrier.Using an Escherichia coli strain overexpressing LovD and a cell-membranepermeable thioester dimethylbutyryl-S-methyl mercaptopropionate(DMB-S-MMP) (FIG. 13A), we developed a whole cell biocatalytic processthat can convert MJ acid to simvastatin acid in one step with highyields (see, e.g., Xie et al., (2007) Appl Environ Microbiol 73:2054-2060). The fermentation process can be an economically competitivealternative to the current synthetic routes.

The thioester DMB-S-MMP is an integral component of simvastatinbioconversion. It is among the most catalytically efficient acyl donorsexamined for the acyltransfer reaction, while being the least expensiveto synthesize. One significant drawback associated with this compound ishydrolysis of the methyl ester bond in DMB-S-MMP that yieldsdimethylbutyryl mercaptopropionic acid (DMB-S-MPA) (FIG. 13). Thehydrolysis reaction is enzymatic because no degradation was observed inthe absence of E. coli, and is dramatically elevated during highcell-density fermentation. The side reaction is undesirable for threereasons: 1) Hydrolysis of the substrate depletes DMB-S-MMP available forLovD-catalyzed transacylation, requiring the thioester to be added inhigh molar excess and to be replenished frequently during fermentation;2) Since DMB-S-MPA is less efficient compared to DMB-S-MMP as adimethylbutyrate donor (˜10 fold slower) (see, e.g., Xie et al., (2007)Appl Environ Microbiol 73: 2054-2060), accumulation of the more solubleDMB-S-MPA can serve as a competing acyl substrate for LovD. Thiseffectively decreases the reaction velocity and has been demonstrated invitro using purified LovD. Therefore, the overall duration of thebioconversion is unnecessarily prolonged; and 3) The carboxylic acidmoiety of DMB-S-MPA interferes with purification of simvastatin acidfrom the culture medium at completion of the bioconversion. Bothcompounds precipitate from the culture broth upon acidification of themedium and additional separation steps are required beforecrystallization of simvastatin. While DMB-S-MPA can be removed bywashing the filtrate with excessive amount of water, an appreciableamount of simvastatin acid were lost during the washing steps, resultingin decrease in overall recovery.

Eliminating the undesirable hydrolysis side reaction can thereforeimprove the economics of the whole cell biocatalytic process and thedownstream purification steps. The most immediate goal is therefore topinpoint the enzyme(s) responsible for the undesirable side reaction.Here, we teach the identification of BioH as the E. colicarboxylesterase that hydrolyzes DMB-S-MMP into DMB-S-MPA. Byconstructing a ΔbioH derivative of the LovD overexpression strain, wecompletely eliminated the competing reaction and further improved therobustness of the whole-cell biocatalytic synthesis of simvastatin acid.

Materials, Strains and Plasmids

Monacolin J and DMB-S-MMP were prepared as described previously (see,e.g., Xie et al., (2007) Appl Environ Microbiol 73: 2054-2060). Allreagents were purchased from standard sources. The BL21(DE3) strain [F−omp hsdSB (rB− mB−) gal dcm λ(DE3)] was obtained from Novagen. The Keiocollection was obtained from the National Institute of Genetics, Japan(see, e.g., Baba et al., (2006) Mol Syst Biol 2: 2006 0008). Thesingle-gene knockout mutants were derived from the BW25113 strain [rrnB3DElacZ4787 hsdR514 DE(araBAD)567 DE(rhaBAD)568rph-1]. WA837 (rB−, mB+,gal met), an E. coli B strain that is restriction-minus andmodification-plus was obtained from The Coli Genetic Stock Center (CGSC)(see e.g., Wood, W. B. (1966). J Mol Biol 16: 118-133). The plasmidspAW31(kanr) and pXK8 (kanr) were derived from pET28a and contain thelovD gene from A. terreus and the bioH gene from E. coli, respectively.

Whole-Cell Based Hydrolysis Assay

The selected 57 mutants from the Keio collection together with BW25113,BL21(DE3) and BL21(DE3)/pAW31 were grown to saturation in a 96-well deepwell plate in 1 mL LB media at 37° C. Neat DMB-S-MMP (5 μL) was added toeach culture with a final concentration of 20 mM. After shaking (20° C.,300 rpm) for 10 hours, each culture was extracted with an equal volumeof ethyl acetate (EA)/1% acetic acid (AcOH). The organic phase wasdried, redissolved in 20 μL acetonitrile (CH3CN), and 1 μL was spottedon a TLC plate (silica gel 60 F254). The TLC plates were developed with20% EA in hexane and visualized with iodine.

Analysis of the compounds were also performed with HPLC using ananalytical C18 column (Alltech Apollo 5u, 150 mm×4.6 mm); lineargradient: 60% CH3CN in water (0.1% trifluoroacetic acid [TFA]) to 95%CH3CN in water (0.1% TFA) over 5 min, 95% CH3CN in water (0.1% TFA) for10 minutes, with a flow rate of 1 mL/min. HPLC retention times (tR) wereas follows: MJ lactone form: 3.40 min; DMB-S-MPA: 3.82 min; DMB-S-MMP:6.80 min; simvastatin lactone form: 8.45 min. Both MJ and simvastatinacids were lactonized before HPLC analysis.

Purification of BioH and Enzymatic Assay

The bioH gene was amplified from E. coli genomic DNA by PCR withflanking restriction sites NdeI and EcoRI using the primers5′-AACATATGAATAACATCTGGTGGCA-3′ (SEQ ID NO: 5) and5′-AAGAATTCTACACCCTCTGCTICAACG-3′ (SEQ ID NO: 6). The gene cassette wasdigested and ligated into pET28a to yield the expression construct pXK8.The E. coli BL21(DE3) strain transformed with pXK8 was grown in LBmedium at 37° C. to an OD600 of 0.5, at which time 0.1 mM IPTG was addedto the culture and expression was performed at 20° C. for 16 hours.Cells were resuspended in Buffer A (50 mM Tris-HCl, pH 8.0, 2 mM DTI, 2mM EDTA) and lysed with sonication. Purification of BioH was facilitatedby the N-terminal 6×His tag and Ni-NTA resin (Qiagen). Nearly pure(>95%) BioH was eluted in Buffer A containing 250 mM imidazole, whichwas buffer exchanged into Buffer A, concentrated, aliquoted and flashfrozen.

The hydrolysis assays were performed at room temperature in 50 mM HEPES,pH 7.9. The thioester substrate DMB-S-MMP was added to finalconcentration between 0.1 mM and 1.0 mM. To facilitate solubilization ofDMB-S-MMP, DMSO was added to a final concentration of 10% for all thesamples. The reaction was initiated with the addition of BioH (0.01 μM)and quenched at desired time points (10, 20, 30 minutes) by adding equalvolume of EA/1% AcOH. The organic phase was separated, dried andredissolved in 20 μL of ACN and analyzed with HPLC. The conversions ofDMB-S-MMP to DMB-S-MPA were quantified by integration of the peaks at234 nm. Comparison of the BioH catalytic efficiency towards the threedimethylbutyryl thioesters were performed at 1 mM substrateconcentration and 10 nM BioH concentration.

P1 Transduction

Following standard protocols (see e.g., Miller, J. H. (1992) A shortcourse in bacterial genetics: A laboratory manual and handbook forEscherichia coli and related bacteria. Cold Spring Harbor, N.Y.: ColdSpring Harbor Press), P1 transduction was used to construct the ΔbioHdeletion mutant of BL21(DE3). Because of the different restrictionsystem between E. coli K strain (BW25113) and B strain (BL21), the Bstrain WA837 (rB−, mB+) was used as an intermediate host fortransduction. The ΔbioH::FRT-kan-FRT marker was first transduced fromJW3375 to WA837 to yield YT0 (WA837 ΔbioH::FRT-kan-FR1). Using BL21(DE3)as a recipient and YT0 as a donor, strain YT1 (BL21 ΔbioH::FRT-kan-FRTλ(DE3)) was constructed. The YT1 strain was transformed with the helperplasmid pCP20 which contains a temperature sensitive replication andthermally inducible FLP gene (see e.g., Datsenko et al., (2000) ProcNatl Acad Sci USA 97: 6640-6645; and Wanner, 2000). Removal of the kanmarker to yield YT2 ((BL21 ΔbioH::FRT λ(DE3)) followed publishedprocedures (see e.g., Datsenko et al., (2000) Proc Natl Acad Sci USA 97:6640-6645; and Wanner, 2000).

PCR was used to verify the genetic changes of YT2. Three primers weredesigned. The primers B1: 5′-TGACGGCTTCGCTATCCCAT-3′ (SEQ ID NO: 7); B2:5′-TACACCCTCTGCTTCAACG-3′ (SEQ ID NO: 8); and B3:5′-GCTGGATTGTTfCGCCGATC-3′ (SEQ ID NO: 9) anneal to the upstream geneyhgA, the 3′ end of bioH that was left intact, and the downstream genegntX, respectively. The expected products for PCR reaction using YT2genomic DNA as template are: B1/B2: 602 bp; B1/B3: 1002 bp. The expectedproducts for PCR reaction using BL21(DE3) genomic DNA as template are:B1/B2: 1271 bp; B1/B3: 1771 bp. The expected PCR products were observedfor each strain.

Whole Cell Biocatalysis

Whole-cell catalytic synthesis of simvastatin acid from MJ acid andDMB-S-MMP were performed as described (see, e.g., Xie et al., (2007)Appl Environ Microbiol 73: 2054-2060). The E. coli BL21(DE3)/pAW31 andYT2/pAW31 strains were cultured side-by-side for comparison. A singlecolony of the freshly transformed strains was used to inoculate a 5 mLLB culture supplemented with 35 mg/L kanamycin and grown overnight at37° C. The next morning, 100 μL of the overnight culture was inoculatedinto 50 mL cultures containing LB broth, F1 minimal medium and F1 mediumsupplemented with 0.15 mg/L biotin. Growth rates were monitored byperiodically measuring the OD600 reading. When OD600 reached ˜0.5, 100μM IPTG was added to the culture and expression of LovD was performed at20° C. for 16 hours. To mimic high density fermentation conditions, thecells were concentrated 10-fold before addition of substrates.Typically, a 14 mL aliquot of the culture was transferred to a 15 mLcentrifuge tube and the cells were collected by centrifugation (4° C.,4000 g, 10 minutes). The cell pellet was gently resuspended in 1316 μlof the supernatant, followed by addition of 84 μl of a MJ acid stocksolution (250 mM in H₂O) (final concentration 15 mM). The concentratedculture was then separated into seven 200 μL samples and 1.2 μL of pureDMB-S-MMP was added to each sample (final concentration 25 mM). Theculture was then shaken at 300 rpm at room temperature. At each timepoint, a total extraction was performed by adding 10 μL, 20% SDS to lysethe cells, followed by liquid-liquid extraction with 500 μL EA/1% AcOH.The organic phase was removed, evaporated, and redissolved in 50 μL ACNfor HPLC analysis. For the BL21(DE3) sample, an additional 1.2 μLaliquot of DMB-S-MMP was added after 12 hours to replenish thehydrolyzed substrates.

Identification of BioH as the DMB-S-MMP Esterase

We first aimed to identify the E. coli enzyme that is responsible forthe observed hydrolysis of DMB-S-MMP into DMB-S-MPA during fermentation.When different acyl carriers such as dimethylbutyryl-S-ethylmercaptopropionate (DMB-S-EMP) and dimethylbutyryl-S-methylthioglycolate (DMB-S-MTG) were used as the thioester substrate, weobserved the corresponding hydrolyzed carboxylic acids in thefermentation broth. This suggests the responsible enzyme is an esteraseor hydrolase with relaxed substrate specificity towards esterfunctionalities.

We used a high-throughput approach to identify the responsible enzyme,utilizing the E. coli K-12 in-frame single gene knockout mutant library(Keio Collection) (see e.g., Baba et al., (2006) Mol Syst Biol 2: 20060008). We reasoned that any mutant strain that is unable to hydrolyzeDMB-S-MMP is directly due to the specific gene deletion. Examination ofE. coli genome annotations (see e.g., Blattner et al., (1997) Science277: 1453-1474) reveals 23 esterases/esterase-like enzymes, 94hydrolases/hydrolase-like enzymes, and 16 acyltransferases (133 totalcandidate genes). Enzymes with confirmed activities and substrates thatare unlikely to be involved in DMB-S-MMP hydrolysis were not examined inthe first round of assays. The list of 57 BW25113 mutant strainsexamined are shown in Table 2.

The mutants, wild type BW21113 and BL21(DE3) were grown to saturation inLB broth in a 96-well plate. We added 5 μL of neat DMB-S-MMP to eachculture (1 mL) and the plate was shaken vigorously for 10 additionalhours at room temperature. The cultures were acidified, extracted withethyl acetate, and the organic phases were analyzed by thin layerchromatography (TLC) using a mobile phase (20% ethyl acetate in hexane)that enabled separation of DMB-S-MMP and DMB-S-MPA (FIG. 14A). The wildtype BW25113 (lane 58) and BL21(DE3) (lane 59) each showed a comparablelevel of substrate hydrolysis and accumulation of DMB-S-MPA. All of themutants examined displayed hydrolytic activity towards DMB-S-MMP, exceptΔbioH (lane 56, strain JW3357). This surprising finding suggests thatBioH (see e.g., O'Regan et al., (1989) Nucleic Acids Res 17: 8004),which is involved in the biosynthesis of biotin (vitamin H), may be thesole enzyme responsible for the observed hydrolysis in vivo. Additionalexamination using HPLC further confirmed that DMB-S-MPA cannot bedetected in the organic extract of the ΔbioH mutant, while nearly 30%were hydrolyzed in bioH+ strains (FIG. 14B).

Verification of BioH Properties In Vitro

To prove that BioH is directly involved in hydrolyzing DMB-S-MMP duringfermentation, we cloned the bioH gene and expressed it as anN-his-tagged protein from BL21(DE3) (see e.g., Tomczyk et al., (2002)FEBS Lett 513: 299-304). The protein was purified to homogeneity usingNi-NTA affinity chromatography with a final yield of 9 mg/L. Thecatalytic properties of BioH towards DMB-S-MMP were assayed and theextent of hydrolysis was measured by HPLC (234 nm). BioH exhibitedMichaelis-Menten kinetics towards DMB-S-MMP and the kcat and Km valueswere determined to be 260±45 sec-1 and 229±26 μM, respectively (FIG.15A).

The activities of BioH towards other dimethylbutyryl thioesters werealso examined using the HPLC assay and are shown in FIG. 15B. Underidentical reaction conditions (1 mM substrate, 10 nM BioH), we observedthat BioH displayed most the potent esterase activity towards DMB-S-MMP.Decreasing the carboxylic acid backbone length by one carbon led to a2.5-fold decrease in the rate of hydrolysis (DMB-S-MTG), whileincreasing the size of the ester moiety to an ethyl ester significantlyattenuated the rate of substrate hydrolysis (DMB-S-EMP). Theseobservations are consistent with the in vivo result, where bothsubstrates were hydrolyzed in the presence of cells, albeit to lesserextents than DMB-S-MMP.

BioH is an essential enzyme in the biosynthesis of biotin in E. coli(see e.g., Lemoine et al., (1996) Mol Microbiol 19: 645-647). It hasbeen proposed that BioH is responsible for synthesizing pimeloyl-CoA(see e.g., Guillen-Navarro et al., (2005) FEMS Microbiol Lett 246:159-165), but its exact biochemical function has not been confirmed.Interestingly, the crystal structure of E. coli BioH has been determinedto 1.7 Å resolution in an effort to predict protein function fromstructural features (see e.g., Sanishvili et al., (2003) J Biol Chem278: 26039-26045). High throughput structural analysis unveiled acatalytic triad in BioH that is also found in known hydrolases, whichhinted BioH may possess hydrolytic activity. Assays using p-nitrophenolesters showed that BioH displays carboxylesterase activities withpreference towards short chain fatty acid esters (see e.g., Sanishviliet al., (2003) J Biol Chem 278: 26039-26045). Our work, both in vivo andin vitro, further elaborates the biochemical properties of BioH andshows that the enzyme has very broad substrate specificity towardsesters moieties. In contrast, BioH displayed no thioesterase activitiestowards the thioester bond present in the substrates analyzed in thiswork. No further degradation of the hydrolyzed thioester acids wereobserved.

Construction of BL21(DE3) ΔBioH Mutant YT2

After identification and verification of BioH as the enzyme responsiblefor hydrolyzing DMB-S-MMP during fermentation, it was evident that aBioH deficient E. coli strain should be used as the host for whole cellbiosynthesis of simvastatin acid. We constructed various expressionvectors of LovD that does not require the T7 polymerase and transformedthem into JW3357. Evaluation of simvastatin acid bioconversion rates inthese strains showed the LovD activity is significantly lower than thatof BL21(DE3)/pAW31. The slower reaction velocities are largelyattributed to the lowered expression levels of LovD in these host/vectorcombinations, as determined by SDS-PAGE. As a result, we concluded thata ΔbioH derivative of BL21(DE3) is needed for achieving maximumconversion rates, while eliminating substrate hydrolysis.

Each of the Keio Collection single-gene knockout mutants contained akanamycin resistance gene in place of the target gene (see e.g., Baba etal., (2006) Mol Syst Biol 2: 2006 0008). The marker is flanked by FRTsites which facilitates facile removal of the marker by the FLP enzyme.We used P1 transduction to move the ΔbioH::FRT-kan-FRT marker fromJW3357 to BL21(DE3). Due to the restriction differences between thedonor K strain and the recipient B strain, we used E. coli WA837(restriction-minus, modification-plus) as an intermediate host for P1transduction (see e.g., Dien et al., (2001) J Ind Microbiol Biotechnol27: 259-264). After transplanting the marker into BL21(DE3) to yieldYT1, the helper plasmid pCP20 which contains a temperature sensitivereplicon and a thermally inducible FLP gene (see e.g., Datsenko et al.,(2000) Proc Natl Acad Sci USA 97: 6640-6645; and Wanner, 2000), was usedto remove of the kan gene to yield YT2 (BL21(DE3) ΔbioH::FRT). PCRanalysis using primers annealing to the upstream and downstream regionsconfirmed deletion of bioH, as well as removal of the kan marker (datanot shown).

YT2 was then cultured in LB medium and the DMB-S-MMP hydrolysis assaywas performed. As expected, the new strain catalyzed no detectablehydrolysis of the thioester and no trace of DMB-S-MPA can be found inthe culture broth. DMB-S-MMP can be nearly quantitatively recovered fromthe saturated culture that had been grown for >24 hours, reassuring thatthe substrate can remain intact through prolonged fermentation usingthis strain.

Whole-Cell Biocatalysis Using YT2

We first examined the viability of YT2 as a host for the whole cellbiocatalytic synthesis of simvastatin acid. The expression plasmid pAW31was transformed into YT2 via electroporation to yield YT2/pAW31.BL21(DE3)/pAW31 and YT2/pAW31 were each grown to OD600 of 0.5, followedby induction of protein synthesis with 100 μM IPTG at 20° C. for up to16 hours. The two strains exhibited identical growth kinetics when LBmedia was used, reaching the same OD600 (3.9-4.0) at the end of proteinexpression period. When F1 minimal medium was used, the two strains grewcomparably before induction (FIG. 16A). In contrast, YT2/pAW31 grewconsiderably slower than BL21(DE3)/pAW31 in medium without biotinsupplementation after induction. The post-induction cell density for themutant strain was ˜60% of the parent strain. We attributed the retardedgrowth rate to the inability of YT2/pAW31 to synthesize biotin andsupport robust cell growth in the minimal medium. When YT2/pAW31 strainwas grown in F1 medium supplemented with 0.15 mg/L biotin, the growthkinetics of the mutant strain were indistinguishable from that ofBL21(DE3)/pAW31.

We then compared the efficiency of YT2/pAW31 to BL21(DE3)/pAW31 in thewhole cell assay. Both strains were grown in LB medium and wereconcentrated ten-fold to mimic a high cell density environment after 12hours of LovD expression. MJ acid and DMB-S-MMP were added to finalconcentrations of 15 mM and 25 mM, respectively, to initiate thebioconversion. FIG. 16B shows that YT2/pAW31 was significantly moreefficient in the assay as a result of bioH deletion. Complete conversion(>99%) was observed for the mutant strain in less than 12 hours ofincubation with no trace of DMB-S-MMP hydrolysis. In contrast,BL21(DE3)/pAW31 achieved the same conversion in 24 hours, whilerequiring addition of another 25 mM of DMB-S-MMP during the fermentationas a result of substrate hydrolysis. During the linear range of thereaction, YT2/pAW31 synthesized simvastatin acid at a rate of 1.5mM/hour, significantly higher than the 0.75 mM/hour measured forBL21(DE3)/pAW31 (see, e.g., Xie et al., (2007) Appl Environ Microbiol73: 2054-2060). Using YT2/pAW31, we were also able to decrease theminimal concentration of DMB-S-MMP required to drive the reaction tocompletion. Complete conversion to simvastatin acid can be achieved withidentical rates when the initial DMB-S-MMP (18 mM) to MJ acid (15 mM)molar ratio was as low as 1.2.

The enhancement in rate is largely due to the increased intracellularconcentration of DMB-S-MMP in the absence of BioH. From the in vitrostudies, we showed that BioH is extremely rapid in hydrolyzingDMB-S-MMP. Therefore, even at low intracellular concentrations of BioH,the substrate hydrolysis reaction can compete for the pool of substratesthat are present in the cytoplasm. Maintaining elevated intracellularconcentration of DMB-S-MMP is critical, especially considering LovD cancatalyze the reversible reaction in which simvastatin acid can behydrolyzed to MJ acid in the absence of acyl thioester donors (see e.g.,Xie et al., (2006) Chem Biol 13: 1161-1169).

Purification of simvastatin acid after bioconversion was also improvedas demonstrated by working up a scaled-up fermentation of YT2/pAW31 (200mL, 15 mM MJ, 18 mM DMB-S-MMP). After verification of completeconversion of MJ acid into simvastatin acid by HPLC (FIG. 17, trace a),the combined fermentation broth and cell extract was washed with hexane,acidified with 6 M HCl and filtered to collect the precipitatedsimvastatin acid (trace b). After washing the filter cake with 1 volumeof dH2O, simvastatin acid was solublized in ACN, filtered and analyzedby HPLC (trace c). No additional washing steps were required to removeDMB-S-MPA that was present in BL21(DE3)/pAW31. The final recovery ofsimvastatin acid using this approach was 94%.

Conclusion

As illustrated above, we have used Keio single-gene knockout library toidentify BioH as the carboxylesterase that hydrolyzes DMB-S-MMP duringwhole cell biocatalytic conversion of simvastatin acid from MJ acid.BioH exhibits very rapid hydrolysis rates, which depletes theintracellular concentration of DMB-S-MMP available as an acyl donor inthe LovD-catalyzed transesterification. Using the bioH expression strainYT2, we were able to completely eliminate degradation of DMB-S-MMP andsignificantly increase the robustness of the whole cell biocatalyst.This strain may be a useful host in other precursor directedbiosynthesis and biocatalysis applications where one or more substratesused contains a labile ester linkage (see e.g., Murli et al., (2005)Appl Environ Microbiol 71: 4503-4509).

Example 4: Efficient Synthesis of Simvastatin Using Whole-CellBiocatalysis

This example describes a one-step, whole cell biocatalytic process forthe synthesis of simvastatin from monacolin J. As discussed in detailbelow, using an Escherichia coli strain overexpressing A. terreus LovD,we were able to achieve >99% conversion of monacolin J to simvastatinwithout the use of any chemical protection steps. A key finding was amembrane permeable substrate,α-dimethylbutyryl-S-methyl-mercaptopropionate (DMB-S-MMP), that wasefficiently utilized by LovD as the acyl donor. The process was scaledup for gram-scale synthesis of simvastatin. We also demonstrate thatsimvastatin synthesized via this method can be readily purified from thefermentation broth with >90% recovery and >98% purity as determined byHPLC. Bioconversion using high cell density, fed-batch fermentation wasalso examined. The whole cell biocatalysis can therefore be anattractive alternative to the current, multistep semisynthetictransformations.

Currently, two semisynthetic processes (see, e.g., Askin et al., 1991,J. Org. Chem. 56; and Hoffman et al., 1986 J Med Chem 29:849-52) arewidely used to synthesize simvastatin starting from lovastatin (FIG.18). One commonly adapted process (see e.g., Hoffman et al., 1986 J MedChem 29:849-52) starts with the hydrolysis of lovastatin to yield thekey intermediate monacolin J, followed by lactonization of the acid toprotect the C11 hydroxyl group, and trimethylsilylation protection ofthe C13 hydroxyl. The protected monacolin J is then subjected toacylation by α-dimethylbutyryl chloride to yield the protected form ofsimvastatin, which is subsequently deprotected to yield simvastatin.Both multistep processes shown in FIG. 18 are laborious, thuscontributing to simvastatin being nearly five times more expensive thanlovastatin. Therefore, a new semisynthetic scheme that can decrease thenumber of chemical transformations and increase the overall efficiencyof the conversion can be of significant utility.

We have previously described cloning and characterization of a dedicatedacyltransferase from the lovastatin biosynthetic gene cluster thatregioselectively transfers the α-methylbutyryl group from the lovastatindiketide synthase (LDKS) to the C8 hydroxyl group of monacolin J toyield lovastatin (see, e.g., Xie et al., 2006, Chem Biol 13: 1161-1169).We demonstrated that LovD has broad substrate specificity towards theacyl carrier, the acyl group and the decalin core. Most notably, LovDwas able to catalyze the direct acylation of monacolin J byα-dimethylbutyryl-S—N-acetylcysteamine (DMB-S-NAC) to affordsimvastatin. The reaction is highly regiospecific towards the C8 alcoholonly, and is therefore a potential one-step process to producesimvastatin from monacolin J without employing protective chemistry.

In this Example, we describe the development of a whole-cellbiocatalytic process that is able to convert monacolin J to simvastatinin a highly efficient manner. Using a novel thioester as the acyl donor,we were able to achieve >99% conversion of simvastatin from monacolin Jin a single step. The fermentation process can be easily scaled up toproduce industrial-scale yield of simvastatin.

Synthesis of α-dimethylbutyryl-S-methyl 3-mercaptopropionate (DMB-S-MMP)

Dimethylbutyryl chloride (1.76 mL, 12 mmol) was added slowly to a 50 mLsolution of methyl-3-mercaptopropionate (1.30 mL, 12 mmol) andtriethylamine (3.340 mL, 24 mmol) in diethyl ether at 0° C. and thesolution was stirred for 2 hours. The reaction was quenched with aqueousNH4Cl and extracted with 100 mL ethyl acetate twice. The organic layeris combined, dried, and evaporated to give a colorless liquid (2.6 g).The residue was purified with silica-gel chromatography (EA/hexane,20/80) to yield pure DMB-S-MMP (2.35 g, 90% yield). 1H NMR: δ 3.87 (s,3H), 3.09 (t, 2H, 7.1 Hz), 2.58 (t, 2H, 7.0 Hz), 1.59 (q, 2H, 7.5 Hz),1.18 (s, 6H), 0.83 (t, 3H, 7.4 Hz); 13C NMR: δ 206.16, 172.26, 51.81,50.04, 34.38, 33.73, 24.71, 23.60, 8.92.

Kinetic Assay of lovD Assay:

Expression and purification of LovD has been previously described indetail (see, e.g., Xie et al., 2006, Chem Biol 13: 1161-1169). Theassays were performed at room temperature in 50 mM HEPES, pH 7.9. Theinitial velocities (ki) of the acyl substrates were determined with 10μM LovD, 1 mM monacolin J and 4 mM of the thioester. The reactionmixture at different time points was quenched by trifluoroacetic acid(TFA), extracted with ethyl acetate, evaporated to dryness andredissolved in acetonitrile. The percent conversion of monacolin J tosimvastatin was determined by HPLC analysis (C18). The linear range ofthe turnover rate is reported as ki in Table 1B. To determine the Km ofDMB-S-MMP, the concentration of monacolin J is fixed at 2 mM, while theconcentration of DMB-S-MMP was varied from 0.2 mM to 10 mM. DMSO isadded to a final concentration of 5% to facilitate solubilization ofDMB-S-MMP. To obtain Km of monacolin J, DMB-S-MMP concentration is fixedat 2 mM, while the concentration of monacolin J is varied from 0.2 mM to5 mM.

Whole Cell Lysate Activity Assay

A whole cell lysate assay was used to determine the level of LovDactivity under different fermentation conditions. For example, the E.coli BL21(DE3) strain transformed with pAW31 was grown in LB medium at37° C. to an OD600 of 0.5, at which time 100 μM IPTG was added to theculture and expression was performed at room temperature (RT) for 16hours. A 70 ml aliquot of the culture was then harvested bycentrifugation (4000 g, 20 minutes). The supernatant was removed, andthe cell pellet was resuspended in 7 ml lysis buffer (20 mM Tris-HCl, pH7.9, 500 mM NaCl). Cells were lysed by sonication on ice and cell debriswas removed by centrifugation (13,000 rpm, 10 minutes, 4° C.). Thelysate is then directed added to the in vitro assay. Final assaycondition: 50 mM HEPES, pH 7.9, 1 mM monacolin J, 4 mM DMB-S-MMP, 5 μlcell lysate, 25 μl final volume. The effective concentration of LovD inthe fermentation broth was then calculated from the initial velocityusing 0.6 min-1 as the turnover rate.

Low-Density Fermentation

The E. coli BL21(DE3)/pAW31 strain was grown in LB medium at 37° C. toan OD600 of 0.5, at which time 100 μM IPTG was added to the culture andexpression was performed at RT for 16 hours. A 10 ml culture wastransferred to a 15 mL centrifuge tube. The cells were collected bycentrifugation (4000 g, 10 minutes). The cell pellet was resuspended in957 μl of the supernatant. The pH was adjusted to pH 7.9 with 1.0 NNaOH, followed by addition of 33 μl 450 mM MJ (final concentration 15mM) and 6 μl pure DMB-S-MMP (final concentration ˜25 mM). The culturewas then shaken at 300 rpm at room temperature. At each time point, a 4μL aliquot was removed from the reaction mixture, quenched in 300 μLethyl acetate containing 1% TFA. The organic phase was removed,evaporated, and redissolved in acetonitrile for HPLC analysis.

For larger scale synthesis of simvastatin, the above procedure wasscaled up starting from 2×1 L shake flask culture of E. coliBL21(DE3)/pAW31 strain. After 16 hours of expression at RT, the culturevolume was concentrated to 200 mL to a final OD600 of 22. Monacolin J(sodium salt form) and DMB-S-MMP were added to a final concentration of15 mM and 25 mM, respectively. The cells were shaken at room temperatureand HPLC was used to monitor the reaction progress. To purify thesimvastatin from fermentation after the reaction has completed (>99%conversion as judged by HPLC), the following procedure was used. Cellswere removed by centrifugation. The intracellular simvastatin wasextracted by stirring the cell pellet in acetone. The acetone was thenremoved under reduced pressure and the residue was dissolved in dH2O andfiltered. The filtrate and the clear fermentation broth were combined,washed with equal volume of n-hexane twice, and acidified to pH 2.0 with6N HCl. The white precipitate was recovered by filtration and was washedexcessively with ice-cold dH2O to remove the coprecipitated DMB-S-MPA.After drying of the filter cake under vacuum, the solids were stirred in200 mL of acetonitrile for 1 hour, followed by filtration to removeinsolubles. The filtrate was evaporated to dryness under reducedpressure to yield the acid form of simvastatin.

High Density F1 Fed-Batch Fermentation

The F1 fed-batch fermentation and media composition were adopted fromPfeifer et al (see e.g., Pfeifer et al., 2002, Appl Environ Microbiol68:3287-92). We excluded the vitamin solution from both the fermentationmedium and the feed medium. A starter culture was grown overnight in 5ml of LB media (with 35 mg/L kanamycin) at 37° C. and 250 rpm and 1 mLwas used to inoculate a 100 ml shake flask seed with F1 medium (with 35mg/L kanamycin). 10 mL of the seed F1 culture was used to inoculate a2-liter Applikon Biobundle vessel containing 1 L of F1 medium.Fermentation was conducted at 37° C. and the pH was maintained at 7.1throughout the experiment with 1 M H2SO4 and half-concentrated NH4OH.Aeration was controlled at 0.2 to 0.4 L/min and agitation was maintainedat 900 rpm. When the OD600 reading reaches between 5 and 10, thetemperature of the fermentation was reduced to RT, followed byadditional of 200 μM IPTG to induce protein expression. At the sametime, a peristaltic pump delivered 0.08 mL/min of the feed solution tothe fermentor.

Effective LovD activity and concentration at different stages of thefermentation were measured as described above. To prepare resting cellsfor bioconversion studies, the cells were centrifuged at 5000 g for 10minutes, followed by gentle resuspension in the same volume of PBSbuffer, pH 7.4. Monacolin J and DMB-S-MMP were then added to the cellaliquots to initiate the synthesis of simvastatin.

Results

Identification of a Kinetically Superior Acyl Donor

We previously showed that LovD can utilize membrane permeable thioestersas acyl donors in the transesterification reaction (see e.g., Xie etal., 2006, Chem Biol 13: 1161-1169). Both DMB-S-NAC and DMB-S-MTG (Table1B) were shown to be substrates of LovD. The two substrates, however,both supported poor turnover in the synthesis of simvastatin, withapparent ki values ˜0.02 per minute. In addition, when either substrateis utilized, the reaction suffered severe substrate inhibition atincreasing concentrations of monacolin J. Therefore, the first priorityin developing LovD into an industrially useful catalyst for thechembiosynthesis of simvastatin is to identify better substrates thatcan overcome these two limitations.

We synthesized several additional variants of DMB-S-MTG and assayed forthe catalytic properties in vitro. DMB-S-methyl mercaptopropionate(DMB-S-MMP), DMB-S-ethyl mercaptopropionate (DMB-S-EMP) and DMB-S-methylmercaptobutyrate (DMB-S-MMB) were each synthesized by reactingα-dimethylbutyryl chloride with the corresponding free thiols. Theinitial turnover rates are compared in Table 1B. Surprisingly,increasing the length of the thioester carrier by one carbon (from C2 inS-MTG to C3 in S-MMP and 5-EMP) significantly increased the turnoverrate of the reaction. Inserting an additional carbon in S-MMB (C4)resulted in additional increase in the rate of transesterification.Under the standard reaction condition (1 mM MJ, 4 mM acyl substrate, 10μM LovD, 50 mM HEPES, pH 7.9), the initial turnover rates ofesterification are 0.6, 0.7, 0.78 min-1 for DMB-S-MMP, DMB-S-EMP andDMB-S-MMB respectively. The >30 fold increases in rates reflect therelative position of the carbonyl groups to the thioester linkage iscritical for binding to LovD. In contrast, when DMB-S-MPA was used asthe acyl donor, the initial turnover rate decreased significantly to0.08 per min, indicating the free acid (likely a sodium salt of) bindspoorly to LovD active site.

We chose DMB-S-MMP as the most suitable acyl thioester for biocatalysisbecause the precursor thiol (methyl mercaptopropionate) is significantlycheaper than the other starting materials. The kinetic parameters of theacylation reaction when DMB-S-MMP is used as the acyl donor weredetermined and are shown in FIG. 19. To obtain the Km values of eithersubstrate in the transacylation reaction, we fixed the concentration ofone substrate, while varying the concentration of the other substrate toobtain a Michaelis-Menten kinetics curve. FIG. 19A shows the reactionturnover rate (V/Eo) as a function of monacolin J concentration. Incontrast to previously assayed substrates, no substrate inhibition bymonacolin J is observed and the Km is determined to be 0.78±0.12 mM. Theturnover rate of LovD at varying amounts of DMB-S-MMP is similarlydetermined by fixing the monacolin J concentration at 2 mM (FIG. 19B).The Km of DMB-S-MMP is shown to be 0.67±0.04 mM. In both titrations thekcat of the reaction is determined to be 0.66±0.03 min-1. Our kineticsanalysis clearly demonstrates that DMB-S-MMP is a kinetically superiorsubstrate compared to previously reported thioesters. The level ofsubstrate inhibition in a Ping Pong Bi Bi reaction depends upon therelative Km of the two substrates. When DMB-S-MMP is used as an the acylsubstrate, its relatively lower Km allows it to bind to LovD readily andis therefore not inhibited by increasing monacolin J concentrations.This important property therefore allows the development of a batchbiocatalytic process in contrast to a fed-batch process in which themonacolin J has to be continuously supplied to keep its concentrationlow and minimize substrate inhibition.

We previously observed that acyl-S-MTG was rapidly hydrolyzed when addedto an E. coli fermentation broth reaction (see e.g., Xie et al., 2006,Chem Biol 13: 1161-1169). To test the stability of the newly identifiedacyl donor under in vivo conditions, neat DMB-S-MMP was added to LBmedium inoculated with E. coli BL21(DE3) cells and the culture was grownat 37° C. overnight (16 hours). We observed ˜20% degradation of thesubstrate to a predominantly more polar compound. Compare to knowncompounds using HPLC, we identified the major degradation product to beDMB-S-MPA, which may arise through the action of endogenous E. colilipases. We concluded that when DMB-S-MMP is supplied in excess in thebatch reaction, the low extent of degradation will not limit thebioconversion of monacolin J to simvastatin.

Whole Cell Biocatalysis

Equipped with the significantly more efficient DMB-S-MMP thioester, westudied the conversion of monacolin J to simvastatin using E. coli as awhole-cell biocatalyst. A BL21(DE3) strain transformed with the pET28aderived expression plasmid pAW31 was used as the microbial host.Expression of LovD was performed for 16 hours at room temperature andthe level of expression was visualized with SDS-PAGE. To quantify theactive amount of LovD expressed under different growth conditions andmedia, we employed an activity assay in which the whole cell lysate wasdirectly added to a reaction assay containing 1 mM monacolin J, 4 mMDMB-S-MMP in 50 mM HEPES, pH 7.9. The conversion of monacolin J tosimvastatin was quantified by HPLC and the apparent concentration ofLovD was estimated using the ki value of 0.6 min-1 (Table 1B). Under lowcell density conditions, LovD is expressed almost three times higherfrom LB medium than F1 minimal medium (Table 3).

We then tested the chembiosynthesis of simvastatin in LB medium. Afterovernight expression of LovD, a 10 mL aliquot of the cells wasconcentrated ten-fold to 1 mL in LB. The concentration step wasperformed to achieve a high cell density environment mimickingfermentation conditions and to obtain a higher effective concentrationof LovD (final concentration approximately 20 μM). The sodium salt formof monacolin J was added to a final concentration of 15 mM (from a stockof 450 mM in water) and neat DMB-S-MMP was added to a final concentrateof 25 mM. The reaction mixture was then shaken rigorously at roomtemperature. Time points were taken periodically to check the conversionof monacolin J to simvastatin (FIG. 20A).

As shown in FIG. 20B, the simple, low density culture of BL21(DE3)/pAW31was a robust whole cell biocatalyst for the synthesis of simvastatin. Aninitial lag phase of 2 hours was observed at the onset of adding the twosubstrates, followed by rapid conversion with a linear reaction velocityof ˜0.73 mM per hour. The reaction rate slows at higher conversions(>95%) as the result of monacolin J depletion. Within 24 hours, 99% ofmonacolin J was converted to simvastatin. Based on HPLC analysis, nodegradation of either monacolin J or simvastatin by cellular metabolismwas observed. We observed ˜20% decrease in the OD600 readings of theculture after 24 hours, suggesting the whole cell process may bemaintained for prolonged catalysis.

We reported that LovD is able to hydrolyze lovastatin into monacolin Jin vitro with a kcat of 0.21 min-1 reaction (see e.g., Xie et al., 2006,Chem Biol 13: 1161-1169). To examine whether we can couple thehydrolytic step together with the acylation step in a singlefermentation run, we assayed the rate of lovastatin hydrolysis byBL21(DE3)/pAW31. Under the same in vivo conditions as above, we observeda slow rate of monacolin J formation. Starting with 0.5 mM lovastatin(sodium salt form), we achieved 91% hydrolysis in 48 hours, with aconversion rate of ˜0.01 mM per hour, nearly 75-fold slower than therate of the acylation reaction. This is in sharp contrast to the invitro kinetic results, in which the hydrolysis rate is only three-foldslower than the acylation reaction. The significant attenuation oflovastatin hydrolysis rate in vivo is likely due to the permeationbarrier of the cell membranes towards the more hydrophobic lovastatin.We attempted to alleviate the membrane permeability barrier by using analternative strain BL21(DE3)/pXXK2, in which LovD is cloned with anN-terminal pelB signal sequence for localization into the periplasmspace. No improvement in the hydrolytic activity is observed for thisstrain, indicating the impermeability of the outer plasma membrane isthe main transport obstacle.

Recovery and Purification of Simvastatin

To access the recovery yield of simvastatin from the whole-cellbiocatalytic process, we increased the scale of the bioconversion to afinal volume of 200 mL. This was achieved by concentrating 2×1 L of theexpression strain in LB medium after overnight expression of LovD. Thesodium salt form of monacolin J and DMB-S-MMP were added to a finalconcentration of 15 mM and 25 mM respectively and the reaction progresswas monitored by HPLC. We observed an identical conversion kinetics inthe larger scale process and >99% conversion was obtained 24 hours afteraddition of substrates. Simvastatin obtained from the chemobiosyntheticroute can be readily purified from the fermentation broth without usingchromatography steps. A centrifugation step was used to separate thecells and the fermentation broth. Intracellular simvastatin wasrecovered by stirring the cell pellet in acetone, followed byevaporation, and redissolving in dH2O. The aqueous solution containingthe intracellular simvastatin was combined with the fermentation brothand was washed with n-hexane to remove DMB-S-MMP. The aqueous solutionwas then acidified with 6N HCl to pH 2.0, which resulted inprecipitation of the free acid forms of simvastatin and DMB-S-MPA. As isknown in the art, free acid forms of simvastatin can be converted tosodium, potassium, ammonium, or any other salt derived from alkalineearth elements or other metallic salts. These salts can then beconverted to pure simvastatin, for example using common methodologiesknown in the art. The DMB-S-MPA contaminant can be removed by filtrationand washing the filter cake with excessive dH2O. The acidified filtratecontained <1% of total amounts of simvastatin recovered. Nearly puresimvastatin can be recovered by washing the filter cake withacetonitrile, followed by evaporation of the filtrate (Final recovery:1.13 g, 90%; final purity as determined by HPLC: 98%).

High Cell Density Fermentation

We explored activity of the whole cell biocatalyst using a high celldensity fermentor. The effective LovD concentration measured from abatch fermentor using TB as the medium and that from a fed-batch runusing F1 minimal medium are shown in Table 3. Using the fed batchprocess adopted from that reported by Pfeifer et al (see e.g., Pfeiferet al., 2002, Appl Environ Microbiol 68:3287-92), we were able to obtaincultures with very high cell densities and LovD activities (FIG. 21A).We were able to achieve ˜1.5 g/L of active LovD expression at an OD600of 75 40 hours after inoculation (FIG. 21A).

Due to the high cost of the starting materials, we were not able toperform a bioconversion using the entire fermentation broth. Instead, wesampled aliquots of the culture at various points during thefermentation run (FIG. 21A). The activities LovD in each of the timepoint were determined using the whole cell lysate assay described inMaterial and Methods. We then performed bioconversion using 1 mL of theculture directly and measured the rate of simvastatin formation(Non-resting cells, FIG. 21B). Alternatively we also examined theproperties of the whole cell biocatalyst after resuspension of the cellpellets in PBS, pH 7.4 (Resting cells). As can be seen from FIG. 21B, ateach time point, high conversion rates can be obtained when the cellsare placed in a resting environment. In addition, the total activity ofLovD in the culture is not linearly correlated to rate of conversion.For example, resting cells from time point 3 exhibited the highest rateof simvastatin conversion at nearly 1 g/L/hour, despite having lesstotal LovD activity compared to cells obtained from time point 4. Theexceptional robustness of these cells were able to complete conversionof 15 mM monacolin J to simvastatin in less than 8 hours.

A key finding in this example is the identification of yet another acylthioester that donates an acyl moiety to the C8 hydroxyl group ofmonacolin J in the presence of LovD acyltransferase, one that issignificantly more efficient in this acylation reaction. We previouslyidentified DMB-S-NAC as a substrate of LovD (see e.g., Xie et al., 2006,Chem Biol 13: 1161-1169). However, the acylation reaction proceeded witha less than ideal turnover, and as a result of weak substrate binding toLovD, the second substrate monacolin J became a competitive inhibitor ofDMB-S-NAC. The latter property is especially detrimental since it forcesthe concentration of monacolin J to be kept at a minimum during thebioconversion. Both DMB-S-MMP and DMB-S-MMB are superior to DMB-S-NAC asacyl donors. The kcat value of DMB-S-MMP is approximately 30 fold higherthrough a subtle structural alteration of the acyl carrier, while the Kmvalue is now comparable to that of monacolin J, hence eliminatingsubstrate inhibition of monacolin J. Furthermore, the 5-MMP thiolprecursor is significantly less expensive compared to S-NAC. Consideringacyl S-NAC thioester are used prevalently in the precursor directedbiosynthesis of natural products (see e.g., Jacobsen et al., 1997,Science 277:367-9), the thioester acyl carriers described in this workmay also find important utility in other engineered biosynthesisapplications.

We attempted to develop an in vitro biocatalytic process for convertingmonacolin J to simvastatin. We reasoned that a crudely purified LovD maybe useful to overcome any transport and purification difficultiesassociated with whole cell fermentation. LovD can be readily fractionedfrom a majority of other cellular proteins by a single 30% ammoniumsulfate precipitation step. More than 98% of the LovD activity measuredfrom whole cell lysate can be reconstituted by resolubilizing theprecipitated pellet in 50 mM HEPES, pH 7.9. However, LovD precipitatesreadily (hours) at high protein concentrations (˜100 μM) and slowly(days) at lower concentrations (˜10 μM) under assay conditions at roomtemperature. Interestingly, the precipitated LovD protein can beresolublized upon dilution into the same buffer and regains nearly allactivity.

When moderate amounts of monacolin J (>5 mM) was used in a batch, invitro process, we were not able to achieve >60% equilibrium conversionof monacolin J into simvastatin, even after prolonged incubation. Thisis likely due to a competing reaction in which simvastatin is hydrolyzedback to monacolin J by LovD. When the simvastatin concentration reachesthe millimolar range, the velocity of the hydrolysis reaction reachesthe maximum value (kcat=0.3 min-1) and therefore significantly impedesthe overall net rate of acylation.

Interestingly, the reverse reaction was not a limiting factor under invivo conditions when the statin concentration was between 10 and 15 mM(4 g/L-6 g/L), as evident in the high conversion (>99%) of monacolin Jinto simvastatin achieved in this study. We reason that after monacolinJ has been converted into simvastatin, multidrug exporters such as theAcrAB-TolC tripartite complex are able to extrude simvastatin from theinner membrane or the periplasm to the medium (see e.g., Murakami etal., 2006, Nature 443:173-9). The impermeability of the E. coli outermembrane prevents reentry of the hydrophobic simvastatin. The morepolar, monacolin J is able to continuously diffuse through the membraneand serve as a substrate of LovD. Together, the E. coli outer membraneand its efflux pumps effectively decreases the intracellularconcentration of simvastatin available for hydrolysis, hence attenuatingthe rate of the undesirable reverse reaction, and maximizing theconversion of the desired reaction. However, the efflux pumps normallyexpressed by E. coli may be overloaded when the substrate concentrationare increased to >20 mM. Under elevated concentrations of monacolin J(and hence simvastatin) and DMB-S-MMP, we were able to achieve a maximumconversion of 85˜90%. Examination of the distribution of simvastatin inthe culture revealed a significant amount (>20%) are localized insidethe cells, hence likely leading to increased levels of producthydrolysis. It is unknown whether the simvastatin were partitioned inthe inner membrane, the cytoplasm or the periplasm. We are currentlyexamining methods to increase the rate of simvastatin export throughoverexpression of selected multidrug efflux pumps (see e.g., Masi etal., 2003, J Chromatogr B Analyt Technol Biomed Life Sci 786:197-205).

In this example, we have developed a highly robust whole-cellbiocatalytic process for the synthesis of simvastatin from monacolin J.Using E. coli as the host, a laboratory scale process capable ofgram-scale synthesis has been implemented. Additionally, astraightforward downstream purification scheme has been devised forfacile recovery and purification of the product. We are currently usingrational and directed evolution approaches to improve the catalyticturnover rates of LovD. With optimization of the LovD properties, aswell as metabolic engineering of the host to improve throughput of theconversion, the chemobiosynthetic route of affording simvastatin can bea competitive and attractive alternative to the synthetic routes shownin FIG. 18.

The present invention is not to be limited in scope by the embodimentsdisclosed herein, which are intended as single illustrations ofindividual aspects of the invention, and any that are functionallyequivalent are within the scope of the invention. Various modificationsto the models and methods of the invention, in addition to thosedescribed herein, will become apparent to those skilled in the art fromthe foregoing description and teachings, and are similarly intended tofall within the scope of the invention. Such modifications or otherembodiments can be practiced without departing from the true scope andspirit of the invention.

TABLES

TABLE 1A Acyl-thioesters as substrates of LovD^([a]) Acyl ThioesterConversion^([b]), Acyl Thioester Conversion, RT Substrate RT (min)^([c])Substrate (min)

7%, 5.1

89%, 4.6

35%, 6.0

35%, 4.2

52%, 6.8

6%, 6.5

87%, 6.8

N.R.^([d])

32%, 8.7

N.R.

7%, 10.6

69%, 7.6

50%, 6.8

58%, 7.6

22%, 7.6

10%, 8.5

52%, 7.8

92%, 6.8

33%, 8.7

70%, 7.6

2%, 7.5

17%, 8.5

TABLE 1B Comparison of initial velocities of thioester substrates.Thioester Substrate Abbreviation Initial Velocity (k_(i), min⁻¹)^(a)

DMB-S-NAC 0.02

DMB-S-MTG 0.03

DMB-S-MMP 0.60

DMB-S-EMP 0.70

DMB-S-MMB 0.78

DMB-S-MPA 0.08 ^(a)Reaction conditions: 1 mM MJ, 4 mM thioestersubstrate, 10 uM pure LovD, 50 mM HEPES, pH 7.9; initial velocity isdefined as the rate of initial turnover in the linear range.

TABLE 2 List of E. coli BW25113 mutants screened in this work. Thenumbers correspond to the TLC lanes shown in FIG. 14A. BioH was shown tobe the sole enzyme responsible for DMB-S-MMP hydrolysis.

TABLE 3 Comparison of protein yield from different expressionconditions^(a) Fermentation Condition LovD Concentration (mg/L)^(b) LBlow density 96 F1 low density 34 TB high density 980 F1 fed-batch highdensity 1500 ^(a)The reported yields represent the highest observedyield under respective conditions. ^(b)The protein yield is estimatedfrom in vitro assay using whole cell lysate as described in Materialsand Methods. The conversion observed is used to estimate the LovDconcentration using a turnover rate of 0.6 min⁻¹.

TABLE 4 Polypeptide Sequence InformationAspergillus terreus LOVD transesterase. Accession AAD34555Kennedy et al. Science. 1999 May 21; 284(5418): 1368-72MGSIIDAAAAADPVVLMETAFRKAVKSRQIPGAVIMARDCSGNLNYTRCFGARTVRRDECNQLPPLQVDTPCRLASATKLLTTIMALQCMERGLVDLDETVDRLLPDLSAMPVLEGFDDAGNARLRERRGKITLRHLLTHTSGLSYVFLHPLLREYMAQGHLQSAEKFGIQSRLAPPAVNDPGAEWIYGANLDWAGKLVERATGLDLEQYLQENICAPLGITDMTFKLQQRPDMLARRADQTHRNSADGRLRYDDSVYFRADGEECFGGQGVFSGPGSYMKVLHSLLKRDGLLLQPQTVDLMFQPALEPRLEEQMNQHMDASPHINYGGPMPMVLRRSFGLGGIIALEDLDGENWRRKGSLTFGGGPNIVWQIDPKAGLCTLAFFQLEPWNDPVCRDLTRTFEHAIYAQYQQG(SEQ ID NO: 1).Penicillium citrinum MlcH transesterase ACCESSION BAC20561Abe et al., Mol. Genet. Genomics 267 (5), 636-646 (2002)MAPSIDVIPTAASTAAGMISDMEAAFKSAVKLKQIPGAVVMARSMNGDIDYTRCFGARTVERDECQRLPPMEIDTPLRLASATKLLTTIMALQCMEQGLVDLDENVNRLLPDLSDMQVLTGFDAAGNAIMRDREGIIKLRHLLTHTSGLSYAFLHPLLQEYMAKGYLKTAEKFGIQSRLAPPAINDPGVEWIYGANLDWAGKLIERATGVDLEEFMQKNICEPLGITDMTFKLQQRPDMLARRSDQTRRNENGSLRYDDSVYFRHDGEECFGGQGVFCGPESYMKVLNSLMKHDGLLLKKDTIELMFQPALDAELEKKMNDHMDTTPHINYGAALPPVMRRNFGLGGIIAMGDLDGHNWRREGSLTFGGGPNIVWQIDPTVGLCTLVVFQLEPWNDPICKDLTRKFEKAMYSQVKCRN (SEQ ID NO: 2).Aspergillus terreus LOVF Polyketide Synthase. AAD34559Kennedy et al. Science. 1999 May 21; 284(5418): 1368-72MTPLDAPGAPAPIAMVGMGCRFGGGATDPQKLWKLLEEGGSAWSKIPPSRFNVGGVYHPNGQRVGSMHVRGGHFLDEDPALFDASFFNMSTEVASCMDPQYRLILEVVYEALEAAGIPLEQVSGSKTGVFAGTMYHDYQGSFQRQPEALPRYFITGNAGTMLANRVSHFYDLRGPSVSIDTACSTTLTALHLAIQSLRAGESDMAIVAGANLLLNPDVFTTMSNLGFLSSDGISYSFDSRADGYGRGEGVAAIVLKTLPDAVRDGDPIRLIVRETAINQDGRTPAISTPSGEAQECLIQDCYQKAQLDPKQTSYVEAHGTGTRAGDPLELAVISAAFPGQQIQVGSVKANIGHTEAVSGLASLIKVALAVEKGVIPPNARFLQPSKKLLKDTHIQIPLCSQSWIPTDGVRRASINNFGFGGANAHAIVEQYGPFAETSICPPNGYSGNYDGNLGTDQAHIYVLSAKDENSCMRMVSRLCDYATHARPADDLQLLANIAYTLGSRRSNFRWKAVCTAHSLTGLAQNLAGEGMRPSKSADQVRLGWVFTGQGAQWFAMGRELIEMYPVFKEALLECDGYIKEMGSTWSIIEELSRPETESRVDQAEFSLPLSTALQIALVRLLWSWNIQPVAVTSHSSGEAAAAYAIGALTARSAIGISYIRGALTARDRLASVHKGGMLAVGLSRSEVGIYIRQVPLQSEECLVVGCVNSPSSVTVSGDLSAIAKLEELLHADRIFARRLKVTQAFHSSHMNSMTDAFRAGLTELFGADPSDAANASKDVIYASPRTGARLHDMNRLRDPIHWVECMLHPVEFESAFRRMCLDENDHMPKVDRVIEIGPHGALGGPIKQIMQLPELATCDIPYLSCLSRGKSSLSTLRLLASELIRAGFPVDLNAINFPRGCEAARVQVLSDLPPYPWNHETRYWKEPRISQSARQRKGPVHDLIGLQEPLNLPLARSWHNVLRVSDLPWLRDHVVGSHIVFPGAGFVCMAVMGISTLCSSDHESDDISYILRDVNFAQALILPADGEEGIDLRLTICAPDQSLGSQDWQRFLVHSITADKNDWTEHCTGLVRAEMDQPPSSLSNQQRIDPRPWSRKTAPQELWDSLHRVGIRHGPFFRNITCIESDGRGSWCTFAIADTASAMPHAYESQHIVHPTTLDSAVQAAYTTLPFAGSRIKSAMVPARVGCMKISSRLADLEARDMLRAQAKMHSQSPSALVTDVAVFDEADPVGGPVMELEGLVFQSLGASLGTSDRDSTDPGNTCSSWHWAPDISLVNPGWLEKTLGTGIQEHEISLILELRRCSVHFIQEAMESLSVGDVERLSGHLAKFYAWMQKQLACAQNGELGPESSSWTRDSEQARCSLRSRVVAGSTNGEMICRLGSVLPAILRREVDPLEVMMDGHLLSRYYVDALKWSRSNAQASELVRLCCHKNPRARILEIGGGTGGCTQLVVDSLGPNPPVGRYDFTDVSAGFFEAARKRFAGWQNVMDFRKLDIEDDPEAQGFVCGSYDVVLACQVLHATSNMQRTLTNVRKLLKPGGKLILVETTRDELDLFFTFGLLPGWWLSEEPERQSTPSLSPTMWRSMLHTTGFNGVEVEARDCDSHEFYMISTMMSTAVQATPMSCSVKLPEVLLVYVDSSTPMSWISDLQGEIRGRNCSVTSLQALRQVPPTEGQICVFLGEVEHSMLGSVTNDDFTLLTSMLQLAGGTLWVTQGATMKSDDPLKALHLGLLRTMRNESHGKRFVSLDLDPSRNPWTGDSRDAIVSVLDLISMSDEKEFDYAERDGVIHVPRAFSDSINGGEEDGYALEPFQDSQHLLRLDIQTPGLLDSLHFTKRNVDTYEPDKLPDDWVEIEPRAFGLNFRDIMVAMGQLESNVMGFECAGVVTSLSETARTIAPGLAVGDRVCALMNGHWASRVTTSRTNVVRIPETLSFPHAASIPLAFTTAYISLYTVARILPGETVLIHAGAGGVGQAAIILAQLTGAEVFTTAGSETKRNLLIDKFHLDPDHVFSSRDSSFVDGIKTRTRGKGVDVVLNSLAGPLLQKSFDCLARFGRFVEIGKKDLEQNSRLDMSTFVRNVSFSSVDILYWQQAKPAEIFQAMSEVILLWERTAIGLIHPISEYPMSALEKAFRTMQSGQHVGKIVVTVAPDDAVLVRQERMPLFLKPNVSYLVAGGLGGIGRRICEWLVDRGARYLIILSRTARVDPVVTSLQERGCTVSVQACDVADESQLEAALQQCRAEEMPPIRGVIQGAMVLKDALVSQMTADGFHAALRPKVQGSWNLHRIASDVDFFVMLSSLVGVMGGAGQANYAAAGAFQDALAEHRMAHNQPAVTIDLGMVQSIGYVAETDSAVAERLQRIGYQPLHEEEVLDVLEQAISPVCSPAAPTRPAVIVTGINTRPGPHWAHADWMQEARFAGIKYRDPLRDNHGALSLTPAEDDNLHARLNRAISQQESIAVIMEAMSCKLISMFGLTDSEMSATQTLAGIGVDSLVAIELRNWITAKFNVDISVFELMEGRTIAKVAEVVLQRYKA (SEQ ID NO: 3).Escherichia coli Carboxylesterase bioH ACCESSION Q8FCT4Welsch et al., Proc. Natl. Acad. Sci. U.S.A. 99(26), 17020-17024(2002)MNNIWWQTKGQGNVHLVLLHGWGLNAEVWRCIDEELSSHFTLHLVDLPGFGRSRGFGALSLADMAEAVLQQAPDKAIWLGWSLGGLVASQIALTHPERVQALVTVASSPCFSARDEWPGIKPDVLAGFQQQLSDDFQRTVERFLALQTMGTETARQDARALKKTVLALPMPEVDVLNGGLEILKTVDLRQPLQNVSMPFLRLYGYLDGLVPRKVVPMLDKLWPHSESYIFAKAAHAPFISHPAEFCHLLVALKQRV (SEQ ID NO: 4).

The invention claimed is:
 1. A method of making simvastatin comprisingthe steps of: (1) combining together monacolin J; an acyl thioester thatdonates an acyl moiety to the C8 hydroxyl group of monacolin J in thepresence of LovD acyltransferase; and a functional LovD acyltransferasethat comprises an amino acid sequence having from 3 to 29 amino acidsubstitution mutations in SEQ ID NO:1; and (2) allowing the functionalLovD acyltransferase to transfer the acyl group from the acyl thioesterto regioselectively acylate the C8 hydroxyl group of monacolin J; sothat simvastatin is made; wherein the acyl thioester is selected topossess at least one of the following properties: (a) is abutyrlyl-thioester, a N-acetylcysteamine thioester or amethyl-thioglycolate thioester; (b) comprises medium chain length(C3-C6) acyl group moieties; (c) is able to cross the cellular membranesof Escherichia coli or Aspergillus terreus cells growing within afermentation media; or (d) is selected from the group consisting ofα-dimethylbutyryl-S-methyl-mercaptopropionate (DMB-S-MMP),dimethylbutyryl-S-ethyl mercaptopropionate (DMB-S-EMP) anddimethylbutyryl-S-methyl thioglycolate (DMB-S-MTG) anddimethylbutyryl-S-methyl mercaptobutyrate (DMB-S-MMB).
 2. The method ofclaim 1, wherein the monacolin J, the acyl thioester, and the functionalLovD acyltransferase are combined in a fermentation media in thepresence of an isolated organism, wherein the isolated organism is atleast one of: (a) Aspergillus terreus that expresses LovD polypeptide ofSEQ ID NO: 1; (b) Aspergillus terreus that does not express LovFpolypeptide of SEQ ID NO: 3; (c) Escherichia coli that expresses LovDpolypeptide of SEQ ID NO:1; or (d) Escherichia coli that does notexpress bioH polypeptide of SEQ ID NO:
 4. 3. The method of claim 1,wherein the acyl thioester isα-dimethylbutyryl-S-methyl-mercaptopropionate, and wherein theα-dimethylbutyryl-S-methyl-mercaptopropionate is present in thefermentation media in a concentration range of 1 mM-100 mM.
 4. Themethod of claim 2, wherein the monacolin J is produced by the isolatedorganism within the fermentation media.
 5. The method of claim 2,wherein the isolated organism is grown under at least one of thefollowing conditions: (a) at a temperature between 25-40° C.; (b) for atime period between at least 4 to at least 48 hours; (c) at a pH between7-8; or (d) in a fermentation media comprising LB, Fl or TB media. 6.The method of claim 1, wherein the method: (a) produces a composition ofmatter comprising 1% or less of the monacolin J that was initiallycombined with the acyl thioester that donates a dimethylbutyryl moietyto the C8 hydroxyl group of monacolin J in the presence of LovDacyltransferase; or (b) results in at least 95% of the monacolin J addedto the combination being converted to simvastatin.
 7. The method ofclaim 1, wherein the acyl thioester isα-dimethylbutyryl-S-methyl-mercaptopropionate, and wherein theα-dimethylbutyryl-S-methyl-mercaptopropionate is present at aconcentration range of 1 mM-100 mM.
 8. The method of claim 1, wherein:a) simvastatin is made in vitro in the absence of an isolated organism,or b) simvastatin is made in vivo in a fermentation media in thepresence of an isolated organism, and the acyl thioester is derived froman exogenous source.
 9. The method of claim 1, wherein the functionalLovD acyltransferase exhibits: a decreased aggregation; an improvedthermal stability; an improved catalytic activity; an improvedk_(cat)/K_(m) value; an improved soluble expression level; or animproved whole cell activity at 25° C. as compared to the LovDpolypeptide shown in SEQ ID NO:1.
 10. The method of claim 1, wherein thefunctional LovD acyltransferase comprises an amino acid sequence havingfrom 4 to 29 amino acid substitution mutations in SEQ ID NO:1.
 11. Themethod of claim 1, wherein the functional LovD acyltransferase comprisesan amino acid sequence having from 5 to 29 amino acid substitutionmutations in SEQ ID NO:1.
 12. The method of claim 1, wherein thefunctional LovD acyltransferase comprises an amino acid sequence havingfrom 6 to 29 amino acid substitution mutations in SEQ ID NO:1.
 13. Themethod of claim 1, wherein the functional LovD acyltransferase comprisesan amino acid sequence having from 7 to 29 amino acid substitutionmutations in SEQ ID NO:1.
 14. The method of claim 10, whereinsimvastatin is made in vitro in the absence of an isolated organism. 15.The method of claim 11, wherein simvastatin is made in vitro in theabsence of an isolated organism.
 16. The method of claim 12, whereinsimvastatin is made in vitro in the absence of an isolated organism. 17.The method of claim 13, wherein simvastatin is made in vitro in theabsence of an isolated organism.
 18. The method of claim 1, wherein theacyl thioester is a butyrlyl-thioester, a N-acetylcysteamine thioesteror a methyl-thioglycolate thioester.
 19. The method of claim 1, whereinthe acyl thioester comprises medium chain length (C3-C6) acyl groupmoieties.
 20. A method of making simvastatin comprising the steps of:(1) combining together monacolin J; an acyl thioester that donates anacyl moiety to the C8 hydroxyl group of monacolin J in the presence ofLovD acyltransferase; and a functional LovD acyltransferase thatcomprises an amino acid sequence having from 3 to 29 amino acidsubstitution mutations in SEQ ID NO:1; and (2) allowing the functionalLovD acyltransferase to transfer the acyl group from the acyl thioesterto regioselectively acylate the C8 hydroxyl group of monacolin J; sothat simvastatin is made; wherein the method: (a) produces a compositionof matter comprising 1% or less of the monacolin J that was initiallycombined with the acyl thioester that donates a dimethylbutyryl moietyto the C8 hydroxyl group of monacolin J in the presence of LovDacyltransferase; and/or (b) results in at least 95% of the monacolin Jadded to the combination being converted to simvastatin.